Many mouse and human tumours express major histocompatibility complex (MHC) class I-associated antigens that constitute targets for syngeneic cytotoxic T lymphocytes (CTL). Genes encoding such ...antigens were isolated from a mouse mastocytoma and from human melanomas by genetic methods. Isolation and characterization of MHC class I-associated peptides has enabled specific anchor residues to be identified that are typical of peptides that bind to distinct class I molecules. Moreover, CTL specific to particular MHC-peptide combinations have been used to identify naturally occurring antigenic peptides in cell extracts and enabled them to be sequenced directly. Most known MHC ligands are of viral origin or are self peptides derived from normal proteins. Here we use total acid extraction and repeated fractionation to isolate and sequence Lewis lung carcinoma (3LL)-specific peptide(s), which shows sequence homology to the connexin 37 protein. Synthetic octamers based on these sequences bind to 'empty' H-2Kb molecules on RMA-S cells, sensitize RMA-S cells to lysis by specific anti-3LL CTL, and induce anti-tumour CTL. The tumour-associated peptide originates from mutated connexin 37 expressed in 3LL.
Abstract
Objective: Since apoptosis is an important contributor to heart diseases in which ischemia and hypoxia are key elements, we tested the hypothesis that hypoxia predisposes neonatal rat ...ventricular myocytes (NRVM) to Fas-mediated apoptosis, by shifting the balance between antiapoptotic and proapoptotic proteins towards the latter. Methods: Normoxic or hypoxic (22 h, 1% O2) cultured NRVM were exposed to recombinant Fas L (rFasL) for 7 h, and apoptosis measured thereafter. Results: Whereas in normoxic NRVM, rFasL did not cause apoptosis measured by the TUNEL assay (4.8±0.5% in control versus 4.5±0.9% in rFasL), in hypoxic cultures rFasL increased the background apoptosis level by 100%. That Fas was functional in normoxic NRVM, despite its inability to mediate apoptosis, was evidenced by the finding that Fas activation increased the diastolic Ca2+i levels measured by Fura 2 fluorescence, and caused arrhythmias. In support of our working hypothesis, hypoxia increased Fas expression by 200% (measured by quantitative Western blot), and the expression of the proapoptotic proteins ARTS and FADD by 323 and 250%, respectively, and decreased the expression of the antiapoptotic proteins ARC and FLIP by 90 and 60%, respectively. Conclusion: By upregulating Fas expression and key proapoptotic proteins, and by downregulating antiapoptotic proteins, hypoxia predisposes ventricular myocytes to Fas-induced apoptosis.
We have known about the existence of killer lymphocytes since 1960, when they were discovered in connection with transplant rejection in vivo. Since then we have uncovered at least five subsets of ...lymphocytes that can kill other cells in vitro, establishing the study of cell-mediated cytotoxicity (CMC) as a major field of immunological inquiry. Berke and Clark summarize the extensive literature based on the study of CMC in vitro. Several important questions about killer cells have now been answered, for example, how they go about destroying other cells. Research ultimately revealed at least three lytic mechanisms available to killer lymphocytes. But do killer cells actually use these mechanisms in vivo? The possible involvement of CMC in transplant rejection, control of intracellular parasites, cancer, autoimmunity, and immune homeostatic regulation is analyzed in detail, yielding some surprising findings, and outlining important questions that remain unanswered. This extensively documented, comprehensive survey of cell-mediated cytotoxicity traces the history of killer lymphocytes from 1960 to the present, providing a definitive resource for specialists and non-specialists alike.
Both the function and regulation of Fas expression in tumours is poorly understood. Our laboratory has reported that cultured, low Fas-expressing tumours undergo massive, yet reversible, ...up-regulation of cell surface Fas expression when injected into mice. The present study was aimed at determining what causes this enhanced Fas expression and whether the newly expressed Fas functions as a death receptor. Newly expressed Fas is indeed capable of inducing apoptosis. Based on our observation that Fas induction is reduced when tumour cells are injected into immune-deficient mice, we propose that Fas up-regulation in vivo involves the host's immune system. Accordingly, Fas up-regulation occurs in vitro when low Fas-expressing tumour cells are cocultured with lymphoid cells. Furthermore ascitic fluid extracted from tumour-bearing mice trigger Fas up-regulation in low Fas expressing tumours. This last finding suggests that a soluble factor(s) mediates induction of Fas expression. The best candidate for this soluble factor is nitric oxide (NO) based on the following observations: the factor in the ascites is unstable; Fas expression is induced to a lesser degree after injection into inducible NO synthase (NOS)-deficient (iNOS⁻/⁻) mice when compared to control mice; similarly, coculture with iNOS⁻/⁻ splenocytes induces Fas less effectively than coculture with control splenocytes; and finally, the NO donor SNAP induces considerable Fas up-regulation in tumours in vitro. Our model is that host lymphoid cells in response to a tumour increase NO synthesis, which in turn causes enhanced Fas expression in the tumour.
The cure of micrometastases following surgery is the major goal of cancer immunotherapy. We have recently isolated tumour-associated antigen (TAA) peptides, MUT 1 and MUT 2, derived from a mutated ...connexin 37 gap-junction protein, from the malignant 3LL-D122 murine lung carcinoma. We now report that synthetic MUT 1 or MUT 2 induces effective antitumour cytoxic T lymphocytes. Peptide vaccines protect mice from spontaneous metastases of 3LL-D122 tumours. Moreover, peptide vaccines reduce metastatic loads in mice carrying pre-established micrometastases. Tumour-specific immunity was primarily mediated by CD8+ T cells. This is the first evidence that peptide therapy may be effective in treatment of residual tumours and provides a rationale for the development of peptide vaccines as a modality for cancer therapy.
Summary
The theory that Fas ligand (FasL)‐expressing tumours are immune‐privileged and can directly counterattack Fas‐expressing effector T lymphocytes has recently been questioned and several ...alternative mechanisms have been proposed. To address this controversial issue, we analysed the impact of FasL‐expressing tumours on in vivo‐primed cytotoxic T lymphocytes (CTLs) and the mechanisms involved. CTLs were obtained from the peritoneal cavity (PEL) after in vivo priming with syngeneic or allogeneic murine tumour cells. We have found that PEL populations undergo Fas‐based apoptotic cell death when co‐cultured with FasL‐expressing tumour cells and that PEL destruction of cognate targets in a 51Cr‐release assay was markedly inhibited by the pre‐exposure to either cognate or non‐cognate tumour cells expressing FasL. Furthermore, cytocidal function of PEL was markedly inhibited by preincubation with FasL‐negative tumour cells, if and only if they were the cognate targets of the CTL; this CTL inhibition involved FasL–Fas interactions. The killing function of ‘bystander’ PELs, reactive to a third‐party target cell, was inhibited by co‐cultivation with PELs mixed with their cognate target. This activation‐induced CTL fratricide was not influenced by the expression of FasL on the cognate target cells. These studies demonstrate the existence of two distinct pathways whereby FasL‐expressing cells inhibit in vivo‐primed FasL‐ and Fas‐expressing CTLs: first, by FasL‐based direct tumour counterattack, and second, by FasL‐mediated activation‐induced cell death of the CTLs, which is consistent with the concept that FasL expression in vivo could play a role in inducing immune privilege.
Our previous studies have shown that intercellular communication mediated by gap junctions is impaired in most tumors as well as in cancer cell lines. However, connexin genes that encode gap junction ...proteins are only rarely mutated in cancer cells. On the other hand, it was reported that mutated Connexin 37 (Cx37) is the origin of shared tumor-associated antigenic octa-peptides (MUT 1 and MUT 2) of two independently derived lung carcinomas 3LL and CMT 64 of mouse origin. Two Cx37 mutations have been implicated: a Cys-54-Gln substitution in FEQNTAQP (MUT 1) and FEQNTAQA (MUT 2); an additional Pro-59-Ala substitution has been proposed in MUT 2. A Cys-54-Gln mutation in both tumors requires three base changes (TGT-to-CAG) to have occurred twice in independently derived tumors. Another complication stems from the fact that Cys 54, which is located in the extra-cellular domain is conserved in all connexins. Due to the important implications that these findings may have regarding the role of gap junctional communication in lung carcinomas as well as in the origin of tumor-associated antigens, we decided to re-examine these mutations. Thus, we PCR-amplified genomic DNA from 3LL and CMT and sequenced the coding region of Cx37 encompassing codon 54. We then analyzed the PCR products by digestion with the restriction enzyme MaeIII, to discern the presence of the putative mutation. Here we have unambiguously demonstrated that clones K
b39.5 (39.5) and D122 of 3LL, and C6 and E9 of CMT 64, previously employed, have only normal Cx37 sequences, including those of codon 54. Therefore, we concluded that Cx37 is not mutated in 3LL and CMT 64 carcinomas.
Previous studies with CTL lines and CTL hybridomas have suggested that functional CD95 (APO-1/Fas)-ligand (CD95L) expression on effector CTLs is a consequence of specific CTL-target recognition and ...TCR triggering of newly transcribed CD95L. Such a control on the expression of CD95L could provide a double safeguard for killing only cognate target cells. Here the regulation of CD95L expression and function was tested in in vivo primed, alloreactive peritoneal exudate CTL (PEL) from perforin-deficient (P0) mice. CD95L-based, PEL-mediated cytotoxicity was blocked by brefeldin A, an inhibitor of intracellular protein transport, but not by the protein synthesis inhibitor emetine, the immunosuppressive drug cyclosporin A, or the DNA transcription inhibitor actinomycin D. CD95L mRNA transcripts in freshly isolated PEL were shown by RT-PCR; CD95L surface expression was evident by staining with Fas-Fc as well as CD95L Abs. Undiminished CD95L expression and cytocidal activity were found in PEL incubated for 48 h in culture, without adding Ag, mitogen, or cytokines. PEL expressed functional CD95L, yet exhibited target cell-specific killing, except when encountering high CD95-expressing cells. The results indicate that PEL use CD95L probably expressed in the Golgi and/or on the cell surface and do not require newly transcribed CD95L upon target cell conjugation. Hence the TCR-triggered recruitment of preformed CD95L, rather than its biosynthesis, controls CD95L-based specific lysis induced by CTL.
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