Analysis of the cDNA encoding murine interleukin (IL) 17 (cytotoxic T lymphocyte associated antigen 8) predicted a secreted protein sharing 57% amino acid identity with the protein predicted from ORF13, an open reading frame of Herpesvirus saimiri. Here we report on the cloning of human IL-17 (hIL-17), the human counterpart of murine IL-17. hIL-17 is a glycoprotein of 155 amino acids secreted as an homodimer by activated memory CD4+ T cells. Although devoid of direct effects on cells of hematopoietic origin, hIL-17 and the product of its viral counterpart, ORF13, stimulate epithelial, endothelial, and fibroblastic cells to secrete cytokines such as IL-6, IL-8, and granulocyte-colony-stimulating factor, as well as prostaglandin E2. Furthermore, when cultured in the presence of hIL-17, fibroblasts could sustain the proliferation of CD34+ hematopoietic progenitors and their preferential maturation into neutrophils. These observations suggest that hIL-17 may constitute (a) an early initiator of the T cell-dependent inflammmatory reaction; and (b) an element of the cytokine network that bridges the immune system to hematopoiesis.

The requirement for target cell nuclei in the two apoptotic death pathways used by cytotoxic lymphocytes was tested using model effector systems in which the granzyme and Fas pathways of target damage are isolated. Mast cell tumors expressing granzymes A and B in addition to cytolysin/perforin lysed tumor target cells about 10-fold more efficiently than comparable effector cells without granzymes. Enucleated cytoplast targets derived from these cells were also lysed with a similar 10-fold effect of granzymes. In contrast to cytoplasts, effector granzyme expression did not influence lysis of red cell targets. The Fas pathway was assessed using the selected cytotoxic T lymphocyte hybridoma subline d11S, which lysed target cells expressing Fas but not those lacking Fas. Similarly, cytoplasts derived from Fas+ but not Fas- cells were also readily lysed by these effector cells. Thus, neither the nucleus itself nor the characteristic apoptotic nuclear damage associated with the two major cell death pathways used by cytotoxic lymphocytes are required for cell death per se.

We studied the coupling of the TCR/CD3 complex to a T cell effector function, namely Fas-based T-cell-mediated cytotoxicity. Encounter or re-encounter with antigen was mimicked by treating 5 d mixed lymphocyte culture cells or T cell hybridomas with anti-CD3 antibody. This TCR/CD3 engagement induced swift expression of Fas-based cytotoxicity in these cells. Induction of Fas-based cytotoxicity was Ca(2+)-dependent, while its execution was not; induction was sensitive to macromolecular synthesis inhibitors, in line with a demonstrable increase of the Fas ligand (Fas-L) message. We also used T cell hybridomas transfected with various constructs to dissect the involvement of distinct components of the TCR/CD3 complex. The cytoplasmic domain of the CD3 zeta chain was able to transduce by itself a signal leading to Fas-L expression, unless there were mutations in its activation receptor homology sequence 1 (ARH-1) motifs. On the one hand, these findings are relevant to signal transduction pathways coupled to the TCR/CD3, and on the other hand, to the involvement of Fas-based T cell-mediated cytotoxicity in various physiological and possibly pathophysiological situations.

Mechanisms of T cell-mediated cytotoxicity remain poorly defined at the molecular level. To investigate some of these mechanisms, we used as target cells, on the one hand, thymocytes from lpr and gld mouse mutants, and on the other hand, L1210 cells transfected or not with the apoptosis-inducing Fas molecule. These independent mutant or transfectant-based approaches both led to the conclusion that Fas was involved in the Ca(2+)-independent component of cytotoxicity mediated by at least two sources of T cells, namely nonantigen-specific in vitro activated hybridoma cells, and antigen-specific in vivo raised peritoneal exudate lymphocytes. Thus, in these cases, T cell-mediated cytotoxicity involved transduction via Fas of the target cell death signal.

At least some long-term in vitro-cultured cytotoxic T cell clones and uncloned cell populations are able, in the presence of Con A, to lyse other cells, to be lysed by other cells, but not to lyse themselves. This as-yet-unexplained result may have implications as to the mechanism of T cell-mediated cytotoxicity.

In this report we describe a variant of the C57BL/6 T lymphoma EL4 (EL4/Mar) which, in contrast to the parental cell line, expresses neither H-2Kb nor beta2-microglobulin (beta2m) but which does express H-2Db detectable by serology and by alloreactive cytotoxic T lymphocytes (CTL). This observation raises the possibility that H-2Db and perhaps other major histocompatibility complex class I molecules are normally not associated with beta2m on the cell surface. In addition, this report is the first to indicated that alloreactive CTL can interact with a beta2m-free class I antigen.

The requirement for D-glucose in T-cell-mediated cytolysis was studied using mouse spleen cells sensitized against alloantigens in vitro. Glucose was required for cytolysis: (a) cytolysis proceeded in a simple buffered salt solution containing Ca++ and Mg++ (low phosphate-buffered saline, LPBS) in the presence but not in the absence of added glucose; (b) 2-deoxy-D-glucose blocked cytolysis. The block by this agent was overcome by excess glucose added as late as 40 min after the inhibitor. This block was not due to inhibition of NADP reduction, since 2-deoxy-D-glucose failed to interfere with the rate of CO2 production by the pentose cycle which we found to be of significant activity in sensitized spleen cells; (c) dialyzed fetal bovine serum (DFBS) in LPBS supported cytolysis in the absence of added glucose. However, 2-deoxy-D-glucose was also inhibitory under these conditions, suggesting that carbohydrate was required here as well. Further results supported the conclusion that DFBS was not acting as a direct source of the required carbohydrate. The relationship between cytolysis, glucose requirement, and provision of energy was studied. As little as 0.1 mM D-glucose in LPBS supported cytolysis. At this glucose concentration, there was no measurable accumulation of lactate in sensitized spleen cells, but Krebs cycle activity was detectable. In 3 mM glucose or above, the range covered by standard tissue culture media, anaerobic glycolysis became a major source of energy in sensitized spleen cells. Consequently, it appears that in standard tissue culture medium, effector cells can generate sufficient energy for cytolysis either by aerobic or anaerobic metabolism. However, the addition of an energy source alone in the absence of glucose was insufficient to support cytolysis in LPBS. Pyruvate in LPBS did not support cytolysis but was shown to be a good substrate for aerobic metabolism in sensitized spleen cells. Glycogenic amino acids and glycerol also failed to support cytolysis. The stage of cytolysis at which glucose is required was investigated. Glucose was necessary for the calcium-dependent lethal hit phase, but not for the cytochalasin A-blockable recognition stage, nor for 51Cr release from injured target cells. Models for the lethal hit process are discussed, which are compatible with the observed requirement for certain hexoses unrelated to their capacity to serve as sources of energy.

Murine thymus derived (T) lymphocytes primed in vivo to mouse 129 (H-2bc) derived H-2-negative F9 embryonal carcinoma cells and rechallenged in vitro with X-irradiated F9 stimulator cells differentiated into anti-F9 cell immune cytotoxic T lymphocytes (CTL). Using CBA mouse derived splenic responder T cells, F9 stimulator cells triggered a primary cytotoxic anti-F9 response. The CTL generated lysed the F9 antigen-positive target cells F9. PCC3 and PCC4, but not the F9 antigen-negative mouse 129 derived PYS tumor cells, nor LPS induced H-2bc blast cells. Mouse 129 anti-F9 cell antisera but not H-2k anti-H-2bc antisera blocked the lytic interaction with F9 target cells. Similarily unlabeled F9 cells but not H-2bc blast cells inhibited the anti-F9 cell cytotoxicity H-2k anti-F9 cell immune CTL were found to be cytotoxic for syngeneic spermatogonia, known to express the F9 antigen. The results suggest not only that CTL can recognize and lyse H-2-negative target cells, but also that CTL precursors can be sensitized against H-2-negative stimulator cells. From the data available it may be inferred that anti-F9 Cell immune CTL recognize the F9 antigen, known to be linked with the T/t locus. Since anti-F9 cell immune CTL lyse syngeneic spermatogonia, the system may be useful to analyze in vitro the induction and effector phase of a T-cell-mediated cytotoxic autoimmune orchitis.