Generation of anti-allogeneic cytotoxic T lymphocytes depends upon a signal, not delivered by tumor cells, that can be provided in trans in the presence of exogenous interleukin-2.

We have reported that, while immune responses were generated initially against CMS5 tumor cells, they were lost with time. In the present work, we asked whether the tumor cells contributed to this by delivering an inhibitory signal or whether a positive signal, delivered in the form of tumor-derived interleukin-2 (IL-2) or the presence of professional antigen-presenting cells (APC) would be sufficient to enable a sustained response. We observed that the presence of tumor cells did not inhibit the generation of anti-(class I) cytotoxic T lymphocytes (CTL), suggesting that the tumor cells were not directly suppressive. In addition, the lack of a response could not be attributed to insufficient levels of IL-2 alone, since even tumor cells that secreted IL-2 failed to stimulate anti-(class I) CTL. Finally, we observed that professional APC were necessary to deliver an essential signal, but only in the presence of IL-2-secreting tumor cells. Thus, CMS5 cells failed to provide essential signals necessary for CTL generation, but were not directly suppressive.

Tumour cell vaccines that secrete interleukin-2 (IL-2) and interferon gamma (IFN gamma) are recognised by T cells while resisting destruction by natural killer (NK) cells.

The inoculation into mice of genetically engineered tumour cells that secrete IL-2 or IFN gamma results in rejection, while unmodified parental tumour cells grow progressively. In vivo studies demonstrated synergy between IL-2 and IFN gamma leading to the rejection of the transduced tumour cells. IL-2 is required for T cell proliferation and differentiation. IFN gamma induced the upregulation of MHC class I molecules that present peptides to CD8+ T cells. Furthermore, IFN gamma can correct defects in antigen processing. Thus, for T cells, IL-2/IFN gamma-secreting double cytokine tumour cell vaccines might serve as class I+ peptide/antigen presenting depots for developing effector cells. In contrast to T cells, NK cells exert spontaneous killing and kill class I+ targets less well than those that are class I-. For this reason, they may actually have a detrimental effect by destroying a class I+ tumour cell vaccine before adequate T cell stimulation occurs. Based upon this rationale, we tested the hypothesis that an unrecognised benefit of increased class I expression by tumour cells in response to IFN gamma secretion would be to enable cytokine-secreting vaccine cells to resist destruction by NK cells. Our results demonstrated that T cells recognised tumour cells secreting IFN gamma better than those secreting IL-2. NK cells, in contrast, were inhibited by tumour cells that secreted IFN gamma, but not by those that secreted IL-2. The findings suggest that, in addition to upregulating adhesion molecules, MHC molecules, and correcting defects in antigen presentation pathways, IFN gamma secretion may protect tumour cell vaccines from early NK-mediated destruction, keeping them available for T cell priming.

IL-2 gene therapy of solid tumors: an approach for the prevention of signal transduction defects in T cells.

The identification of tumor-associated antigens has focused attention on the mechanisms that underlie the failure of T cells to destroy tumor cells. A deeper understanding of the process of signal transduction following the binding of ligand by the T cell receptor can help to identify underlying defects that may be involved. Gene therapy using tumor cells genetically modified to express cytokines or surface determinants is a promising technique for stimulating antitumor responses. A potential pitfall in its application to cancer, however, is that some patients' T cells are immune suppressed and may resist stimulation by such genetically engineered vaccines. Recent studies have demonstrated that T cells from tumor-bearing patients exhibit abnormalities in signal transduction events, possibly rendering them unable to respond to activation signals. Gene therapy with interleukin 2 secreting tumor cells in an animal model has been shown effective in preventing the onset of signaling defects. A more precise definition of the molecular mechanisms that enable cytokine-secreting tumor cells to stimulate specific antitumor responses may make it feasible to optimize immunotherapeutic approaches resulting in better clinical results.

The impact of gene therapy on T cell function in cancer.

Studies of signal transduction by T cells are slowly identifying the intracellular messengers that must be generated for full T cell activation to take place. The recent, convincing identification of several tumor-associated antigens (TAA) has transformed our task into trying to define the mechanisms that underlie the failure of T cells to destroy antigenic tumor cells. Although there are a variety of hypotheses that explain why tumors grow progressively, even if they are antigenic, recent evidence suggests that T cells from tumor-bearing patients exhibit abnormalities in signal transduction that render them unable to respond to appropriate activation signals, even following proper stimulation. Gene therapy with interleukin-2 (IL-2)-secreting tumor cells in an animal model has been effective in preventing the onset of these signaling defects. Discovery of the molecular mechanisms by which such cytokine-secreting tumor cells induce immune responses and how they may best be applied clinically may provide clearer indications of the directions to pursue to alter the balance between the T cell and the tumor cell in the patient's favor.

Abnormal signal transduction by T cells of mice with parental tumors is not seen in mice bearing IL-2-secreting tumors.

There is considerable evidence to demonstrate that immune function is abnormal in tumor-bearing mice, perhaps accounting, at least in part, for progressive tumor growth. In an attempt to generate an antitumor response, we used retroviral vectors to express IL-2 cDNA in CMS5, a murine fibrosarcoma. Mice inoculated with unmodified tumor cells suffered progressive tumor growth, whereas tumors secreting IL-2 were rejected or grew slowly. Animals bearing unmodified but not IL-2-secreting tumors also were immunosuppressed. On the basis of these observations, we were interested in how IL-2 secretion by the tumor cells prevented the onset of hyporesponsiveness. To identify biochemical differences between T cells of mice with parental vs slowly growing IL-2-secreting tumors, we examined signal transduction after activation through the CD3/TCR complex. Protein tyrosine phosphorylation was altered and calcium flux was reduced in cells of mice with parental tumors compared with animals with slowly growing IL-2-secreting tumors. In addition, levels of protein for the tyrosine kinases p56lck and p59fyn, as well as the TCR-zeta-chain, were reduced. These differences in signal transduction were observed for T cells of mice with parental and IL-2-secreting tumors of the same size, demonstrating that differences in tumor size alone could not explain our findings. Thus, IL-2 secretion by tumors seems to be able to prevent immunosuppression by maintaining normal signal transduction in T cells, facilitating the generation of antitumor responses.

Human tumor vaccines and genetic engineering of tumors with cytokine and histocompatibility genes to enhance immunogenicity.

Genetically engineered tumor cells can be used as vaccines in order to stimulate an immune response. To date, tumor cells have been modified in vitro so that they secrete cytokines or express histocompatibility molecules that they naturally fail to express. These tumor cells differ in the types of immune responses they induce and in whether the responses have local or systemic efficacy. Many questions have been raised during the past year, including whether allogeneic or autologous tumor cells should be employed and whether there may be a risk of inducing autoimmune disease along with the antitumor response. Nevertheless, because of the paucity of available therapies for patients with advanced cancer, investigators must attempt to refine the approaches used in order to minimize patient risk while maximizing tumor cell destruction.

Vaccination with IL-2-secreting tumor cells stimulates the generation of IL-2-responsive T cells and prevents the development of unresponsiveness.

Infection of CMS5 tumor cells with retroviral constructs containing interleukin-2 (IL-2) cDNA and selection in medium supplemented with G418 resulted in the isolation of clones which secreted IL-2. Whereas injection of parental tumor cells resulted in progressive tumor growth, tumor cells secreting high levels of IL-2 were rejected. Furthermore, in animals vaccinated with IL-2-secreting cells, the immunosuppression associated with the inoculation of parental tumor cells did not develop, and these animals resisted a challenge with viable tumor cells. To better understand the functional differences in the anti-tumor responses of immune and tumor-bearing mice which are at the basis for these diverse responses, we used an in vitro model to analyze interactions between splenic lymphocytes and tumor cells. Spleen cells isolated from either tumor-bearing or immune mice proliferated vigorously when cultured alone for 6 days, but much less in the presence of parental tumor cells. This effect could not be transferred with supernatant from tumor cell lines. Spleen cells from tumor-bearing mice remained unresponsive, while those from immune mice proliferated well in response to IL-2-secreting tumor cells. Only spleen cells from immune animals were able to develop cytotoxicity against CMS5 cells following in vitro restimulation. These results are consistent with the interpretation that exposure to parental tumor cells inhibited cell-mediated anti-tumor responses by a mechanism that involved cell-to-cell contact.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor-bearing animals contain suppressed antitumor effectors the function of which can be unmasked by IL-2.

CMS5 fibrosarcoma cells were infected with retroviral constructs containing interleukin-2 (IL-2) cDNA and selected in G418. Parental tumor cells and those that produced IL-2 were injected in vivo. Whereas injection of parental tumor cells resulted in progressive tumor growth, those secreting high levels of IL-2 were rejected. Furthermore, the immunosuppression associated with inoculation of parental tumor cells was not seen. To understand the failure of mice to reject non-IL-2-secreting tumor cells, functional responses of spleen cells from immune and tumor-bearing mice were studied in vitro. As expected, immune spleen cells proliferated under a variety of conditions but were inhibited in the presence of parental tumor cells. Even spleen cells from tumor-bearing animals responded well in the absence of parental tumor cells or in the presence of parental tumor cells, if supplied with adequate levels of IL-2. These results suggest that both tumor-bearing and immune mice generate antitumor effectors but that the cells might be functionally suppressed because of their inability to secrete IL-2 after contact with parental tumor cells.

Post-transcriptional regulation of MHC class II expression in human T cells.

Human T lymphocytes are among those cells which are cell surface class II- in the resting state, but can be induced to express class II following treatment with appropriate stimulators. Although resting T cells do not express detectable surface class II, cell surface class II can be detected on purified T cells as early as 30 min following stimulation with PHA and PMA, well before the initiation of DNA synthesis, and the percentage of positive cells gradually increases with time. One hypothesis explaining this very rapid surface expression of class II is that the genes can be regulated post-transcriptionally in T cells. To test this, we used nuclear run-on assays to measure the transcriptional rate of diverse class II genes in resting and activated T cells. Our results demonstrated that transcripts for DR, DP, and DQ could be detected in cells which were neither dividing nor transcribing mRNA for another marker of T cell activation, the IL-2 gene. Northern blot analysis demonstrated low to moderate steady-state levels of DR beta mRNA in these cells. Moreover, treatment of activated T cells with cycloheximide resulted in superinduction of class II for DR, DQ, and DP. These results suggest that resting T cells can transcribe mRNA for class II genes, but that they do not express the protein product on the cell surface in a detectable way until following activation. In addition, they suggest that there may be a protein factor which negatively influences class II levels in T cells. Thus, the regulation of class II in T cells is complex and involves post-transcriptional regulation, at least in part.

The control of class II expression on T cells is independent of the regulation of Tac and the induction of proliferation.

In order to define the association between class II expression and other markers of T cell activation we tested the ability of various modes of stimulation to induce the expression of class II, Tac, and to stimulate proliferation. Stimulation of T cells with phytohaemagglutinin (PHA) in the presence of accessory cells strongly induced proliferation, Tac and the class II antigen DR. When purified T cells without accessory cells were stimulated with the phorbol ester, PdB, and the calcium ionophore, ionomycin, strong proliferation and Tac expression were induced, but only low levels of surface class II were observed. In contrast, stimulation of the same cells with PHA resulted in weak proliferation, strong Tac, but again low class II levels. The addition of PdB to the PHA increased the proliferative response, but did not affect Tac expression, which remained high, or class II expression, which remained low. Subsequent culture in conditioned medium of purified T cells which had been activated with either PdB and ionomycin or with PHA resulted in increased surface class II levels in both cases. Additional experiments suggested that neither IL-2, IL-4, nor interferon-gamma (IFN-gamma) alone was responsible. These results demonstrate that class II expression can be separated from the induction of proliferation and the upregulation of Tac and that the mode of T cell stimulation influences the resulting activation pathway. Furthermore, they suggest that the control of class II expression on T cells is more tightly regulated than it is on other cells.

The response pattern of human T cells to stimulation following expansion in IL-2.

The ability to stimulate and induce responses of T cells is influenced by their age and state of differentiation. We have examined the response upon restimulation of T cells expanded in IL-2. Our results demonstrate that large numbers of T cells, which after activation react by secreting IL-2, can be obtained from small numbers of PBL. In this study T cells were grown in IL-2 and, after 12-14 days, IL-2 was withdrawn in order to deprive them of growth factors. Our findings showed that after resting for 48 hours without IL-2, IL-2-dependent cells reverted to small lymphocytes, ceased to incorporate 3H-TdR, and had no mRNA for activation antigens such as Tac or IL-2. The cells could be reactivated to proliferate by stimulation with a calcium ionophore ionomycin and phorbol dibutyrate (PdB). Cells from insulin dependent diabetes mellitus patients with a defined immunoregulatory defect were then studied. Our results demonstrated that the IL-2 expanded cells evidenced the immunoregulatory defect for IL-2 synthesis that we had initially defined using virgin T cells from peripheral blood. These results demonstrate that studies of immune function can be undertaken in donors from whom limited numbers of peripheral blood lymphocytes are available using primed cells which have been allowed to dedifferentiate.

Low interleukin-2 synthesis by type 1 diabetics is regulated at the pretranslational level.

Insulin-dependent diabetes mellitus (IDDM) is believed to be a consequence of an autoimmune attack on beta cells by T cells. We have previously reported that T cells from the majority of patients with IDDM produced decreased levels of interleukin-2 (IL-2) following activation with phytohemagglutinin. In this study we began to characterize the basis for this defect. First, we tested whether the decreased IL-2 synthesis was due to the secretion of a factor which inhibited the ability of indicator cells to respond to IL-2 or which inhibited IL-2 release by responder cells. Second, we examined steady-state levels of IL-2 mRNA in IDDMs and controls. To make this feasible, given the limited number of peripheral blood lymphocytes (PBL) available from our patients, we depended upon an approach which enabled the generation of large numbers of resting T cells, called G0/G1 cells, from small numbers of PBL. Preliminary experiments demonstrated that G0/G1 cells from IDDMs reproduced the IL-2 defect seen originally with PBL. Our results suggested that the secretion of inhibitory factors could not explain the IL-2 defect. A comparison of steady-state levels of IL-2 mRNA from activated T cells of IDDMs and age-matched controls, however, demonstrated lower levels of IL-2 mRNA in IDDMs compared to controls. Finally, we observed that the IL-2 mRNA in IDDM T cells was less stabile than that in the control cells, suggesting a possible mechanism for the defect.

Negative signal transmission through class II molecules on activated T cells.

Major histocompatibility complex class II antigens are expressed on human T cells following activation, but their functional role remains obscure. We have investigated the effect of anti-class II monoclonal antibodies on T cell proliferation. Our results indicated that antibodies directed against either DR, DQ, or DP were able to decrease 3H-TdR uptake if the cells had been activated by interleukin-2 (IL-2). On the other hand, minimal inhibition resulted when phorbol dibutyrate, a phorbol ester, and ionomycin, a calcium ionophore, were used to activate cells. The specificity of the effect was demonstrated by the observation that anti-class I antibodies inhibited proliferation stimulated by IL-2 and phorbol dibutyrate and ionomycin equally well. Proliferation by the anti-class II monoclonal antibodies was inhibited regardless of whether the monoclonal antibodies were added at the initiation of culture or to actively proliferating cells, suggesting that an early event was not specifically targeted. Our findings are consistent with the interpretation that class II antigens are involved in the transmission of signals to activated T cells.

Susceptibility of human neuroblastoma cell lines to cytotoxic T lymphocyte-mediated lysis.

The susceptibility of neuroblastoma cells to cytotoxic T lymphocyte (CTL)-mediated killing was investigated. Cytotoxic lines were generated by sensitizing peripheral blood lymphocytes against two stimulator cells, a neuroblastoma line, CHP-100, and normal allogeneic lymphocytes, LS. LS cells shared class I antigens with CHP-100, but in addition expressed class II antigens. The resulting cell lines strongly lysed both CHP-100 and LS cells, but poorly killed the natural killer (NK) target K562. Specific blocking of lysis by a monoclonal antibody directed against class I determinants and strong killing by the line following depletion of cells with NK or LAK markers demonstrated that this neuroblastoma line was lysed by CTL.

The development of cell mediated immunity: very young children have strong LAK activity.

We were interested in evaluating immune function in very young children with cancer who were treated with gamma-interferon on a sequential basis. Though gamma-interferon was reportedly able to enhance NK activity, and while many tumor cells are susceptible to lysis by these cells, this effector mechanism is not fully developed in very young children. Since LAK cells also have anti-tumor activity and are produced in response to stimulation with Interleukin-2, we investigated whether LAK killing might be more readily demonstrable in very young children. We report that LAK activity in this group did not differ significantly from that of adults. This was also true for a small group of neuroblastoma patients tested. Furthermore, as opposed to NK activity, LAK activity was demonstrable following freezing and thawing of PBL.

HLA class II mRNA accumulation by activated human T cells following growth in conditioned medium.

The expression of HLA Class II beta genes by seven CD4- or CD8-positive T cell clones grown in conditioned medium was examined on the mRNA and cell surface levels. Monoclonal antibodies directed against Class II glycoproteins demonstrated the presence of these molecules on the cell surface. In addition, Northern blot analysis demonstrated that cDNA probes for the DR, DP, and DQ beta genes hybridized to mRNA isolated from each clone. Because we were interested in differences in expression of relative levels of DR, DQ and DP, mRNA isolated from the clones was quantified by means of densitometry. Our results demonstrated that different ratios of DR, DQ and DP beta chain messages were expressed by the clones. Subsequent studies established the kinetics of the accumulation of DR beta mRNA messages by T cells during culture and showed that it could be regulated by factors contained within conditioned medium. Our results confirm that the accumulation of Class II mRNA following growth of T cells in conditioned medium appears to be a general feature.

Regulation of HLA-DR, DP, and DQ expression in activated T cells.

Class II expression following restimulation of a population of T cells which had first been activated and then allowed to revert to small resting cells via IL-2 deprivation was followed on the cell surface and mRNA levels. Our interest was to determine the kinetics of class II expression during in vitro growth, the triggers which can induce them, and also whether similar or different patterns are observed for the three class II antigens, DR, DQ, and DP. The cells responded rapidly to restimulation with conditioned medium containing IL-2 as assessed by increased incorporation of [3H]TdR into DNA. Cell surface expression of HLA-DR reached peak levels by Day 1, and greater than 90% of the cells continued to express DR until Day 4, when the number of positive cells gradually began to decline. The expression of DP also increased, though maximum levels were not reached until Day 3, when it began to decline. The percentage of cells positive for DP was, however, consistently lower than that of DR. DQ was expressed by very few cells, but appeared to increase until Day 4 and then decline. Tac expression was dramatically up-regulated following IL-2 stimulation, remaining high until Day 4, and then declining more precipitously than any of the other antigens. Class I antigen expression was relatively constant during the entire culture period, though a slight decline was noted between Day 5 and 6 when proliferation and viability were at their lowest levels. On the molecular level, DR beta mRNA accumulation peaked at Day 3, then rapidly declined. Reculture in IL-2 on Day 4 resulted in transient reaccumulation of message. Study of DP beta and DQ beta mRNA demonstrated strong expression on Days 3 and 4 and no obvious up-regulation after restimulation with IL-2 on Day 4. The kinetics of Tac mRNA accumulation and the response to restimulation with IL-2 closely resembled that of DR. Finally we compared the ability of different signals to up-regulate class II mRNA and surface expression and to restimulate proliferation. Our results indicated that PHA was more effective then medium alone, but far less effective than was IL-2. PMA was essentially no different than medium alone.

The detection of human minor alloantigens following restriction determinant implantation.

The recognition of minor alloantigens by cytotoxic T lymphocytes (CTL) serves as a model for the recognition of tumor and viral antigens. Progress in this area has been limited, however, since CTL recognize minor alloantigens only in association with self-class I antigens. Thus, experiments designed to study minor alloantigens are limited to target cells that share HLA determinants with the CTL. We raised CTL lines that recognized human minor alloantigens. In order to circumvent the problem that only target cells which expressed the appropriate restriction determinants could be tested for minor antigens. Sendai virus mediated fusion was used to integrate appropriate HLA antigens into cells that did not express them naturally. The target cells were then tested in CML for their expression of minor antigens. The results of experiments demonstrated that, following class I implantation, the detection of minor antigens on certain restriction determinant negative cells was possible. Furthermore, the restriction determinant was able to associate with the minor antigen in a manner appropriate for recognition by the T-cell receptor.

Expression of class II antigens by subsets of activated T cells.

Gene products coded for within the HLA complex play an important role in the control of immune responses. Class I antigens, coded for by the HLA-A, B, and C loci, are expressed by virtually all mononuclear blood cells. Class II antigens, coded for by the DR, DQ, and DP loci, have a more limited tissue distribution. They are expressed by B cells, monocytes, and by activated, but not by resting, T cells. The class II molecules of B cells and antigen-presenting cells have long been of interest to immunologists, since they are involved in the presentation of antigen, in communication between T cells and B cells and between T cells and adherent cells, and in susceptibility to certain diseases. The class II antigens expressed by activated T cells, however, remain largely uncharacterized in terms of their specificity, functional significance, and molecular nature. We have studied the expression of DR and DQ antigens by activated T cells and then examined the expression of DR versus DQ antigens by Leu 2a and Leu 3a subsets of mitogen-activated populations. Our results demonstrated that, as for class II-positive macrophages, the intensity of staining with monoclonal antibodies directed against DR antigens was much greater than that obtained with those directed against DQ antigens. Interestingly, the percentages of Leu 2a- and Leu 3a-positive cells which expressed DR antigens were quite similar, as were the percentages of Leu 2a and Leu 3a cells which expressed DQ. Thus, there does not seem to be preferential expression of DR versus DQ antigens by mitogen-activated T-cell subsets. Finally, though both DR-positive-DQ-positive and DR-positive-DQ-negative populations were detected, few or no DR-negative-DQ-positive cells were observed in these populations.

Generation of antigen specific cytotoxic T lymphocytes (CTL) directed against class II molecules expressed by activated T cells.

Mitogen-activated T cells were used to provide a source of Class II antigens to CTL originally stimulated against mononuclear cells expressing both foreign Class I and Class II determinants. Our results indicated that 7-10-day-old activated T antigen-presenting cells, which shared only Class II antigens with the original priming cell, were able to stimulate the differentiation of CTL-recognizing Class II determinants. The use of 14-day-old activated T cells as target cells in the CML assay, compared with 72 h PHA blasts or 7-day-old activated T cells, enabled a more sensitive detection of the anti-Class II CTL.