Isolation of genes overexpressed in freshly isolated breast cancer specimens.

A number of approaches have been used to identify genes important in breast cancer. In one approach the genes already shown to be involved in other tumors, such as p53 and Her2neu, were examined. A second approach examined genes detected through genetic screening of families with a high incidence of breast cancer, for example, BRCA-1 and BRCA-2. We used a third approach, subtractive hybridization, to identify and clone genes that were preferentially expressed in breast cancer cells compared to normal mammary epithelium. Instead of analyzing breast cancer cell lines, we examined fresh human breast cancer specimens. By subtracting normal mammary epithelial cDNA from breast cancer cDNA, we were able to clone several genes overexpressed in breast cancer. Two of these genes, L19 and MLN70, were previously reported to be overexpressed in breast cancer. Three of these genes, L19, L34, and MLN70, were localized to a region on chromosome 17 where Her2/neu and BRCA-1 are found. In addition, we isolated a gene we call breast cancer associated gene-1 that was expressed almost exclusively in fresh breast cancer tissue and not in normal mammary epithelium or breast cancer cell lines. We were unable to detect expression of breast cancer associated gene-1 in cell lines from melanoma, renal cell carcinoma, lymphoma, or leukemia. The full-length sequence from two separate breast cancer specimens revealed one amino acid difference compared to the sequence from normal breast epithelial tissue. Further studies are necessary to determine whether these genes contribute to breast cancer development or can be used as therapeutic targets.

Peripheral T lymphocytes from women with breast cancer exhibit abnormal protein expression of several signaling molecules.

We examined signaling molecules of peripheral blood T lymphocytes obtained from women with breast cancer. In 6 of 14 patients, T lymphocytes displayed an impaired ability to translocate NFêB p65 (Rel-A) following activation by anti-CD3 and IL-2. This observation was made despite normal cytoplasmic levels of the Rel-A protein. We also detected abnormally low levels of the signaling molecules T-cell receptor (TCR)-zeta, ZAP-70 and p56lck in 4 of 14 breast cancer patients, i.e., defects in T-cell signaling molecules. T lymphocytes from 6 of the 14 patients also exhibited an increased expression of the dual specificity phosphatase, map kinase phosphatase-1 (MKP-1). MKP-1 inactivates MAP kinase and therefore may interfere with the activation of c-jun and c-fos. Abnormalities of I or more signaling molecules were found in 9 of 14 patients; however, only 3 patients had T cells that exhibited all 5 defects. Our data have implications for the detection of potentially dysfunctional T cells in patients with cancer. For example, the analysis of only 1 signaling molecule may allow patients with significant defects in T-cell signaling to go unnoticed. Finally, despite impaired Rel-A translocation, T cells were capable of transcribing IL-2. Impairments in the translocation of Rel-B and c-Rel further suggest that the NFKB family members Rel-A, Rel-B and c-Rel are not required for the transcription of IL-2 in the peripheral T lymphocytes of patients with breast cancer.

Adoptive immunotherapy with tumor-infiltrating lymphocytes and interleukin-2 in patients with metastatic malignant melanoma and renal cell carcinoma: a pilot study.

PURPOSE: The objective of this study was to determine the tolerance and effect of moderate-dose recombinant human interleukin-2 (rHu IL-2) and tumor-infiltrating lymphocytes (TIL) in patients with metastatic melanoma (MM) or renal cell carcinoma (RCC) refractory to standard therapy. PATIENTS AND METHODS: Twenty-six patients (18 MM and eight RCC) were entered onto this pilot study. TIL were isolated from fresh biopsy material and activated with anti-CD3 antibody, OKT3, for 48 hours and expanded in 100 IU/mL r-methionyl Hu IL-2 alanine 125 (r-met Hu IL-2 [ala-125]). At least 10(10) TIL were reinfused intravenously in three divided injections on days 2, 4, and 6 of the protocol. A maximum dose of 30,000 U/kg of IL-2 per injection was administered every 8 hours from day 2 through day 11 for a total of 28 doses. RESULTS: Sixteen melanoma patients completed the study. Of these, three (19%) showed a durable complete response (CR), nine (56%) had no response (NR), and four (25%) had progressive disease (PD). One nonresponder demonstrated complete tumor regression within 1 year of treatment. Of four assessable RCC patients, two experienced a minor response (MR) and two showed NR. All TIL cultures showed comparably high cytotoxic activity as determined by antibody-redirected lysis (ARL). More importantly, melanoma TIL from responders possessed significantly higher cytotoxicity against autologous tumor cells than TIL from nonresponders (P < .05). Production of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-gamma), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and IL-4 was similar for TIL from melanoma responders and nonresponders, or TIL from RCC patients. CONCLUSIONS: Immunotherapy with polyclonally activated TIL and moderate-dose IL-2 could be successfully used for the treatment of immunogenic tumors with less toxicity and lower costs as compared with high-dose IL-2 protocols.

T cell receptor V beta 2 and V beta 6 mediate tumor-specific cytotoxicity by tumor-infiltrating lymphocytes in ovarian cancer.

The interaction between T lymphocytes and the Ag-HLA complex on tumor cells is mediated by the TCR. The diversity and the specificity of the TCR are in part secondary to the gene rearrangement of the V region on the beta-chain (V beta). To determine whether a restricted number of TCR V beta genes are utilized in the recognition of ovarian cancer, tumor-infiltrating lymphocytes (TIL) were isolated from six consecutive untreated ovarian cancer patients. TIL were also cultured using repeated autologous tumor stimulation, and by 7 wk, five of six patients produced bulk cultures consisting of > 50% CD8+ T cells and demonstrating an autologous tumor-specific pattern of cytotoxicity. TCR V beta gene usage was analyzed in the five patients yielding fresh TIL and corresponding 7-wk cultured, tumor-specific TIL; 22 primers specific for 20 TCR V beta gene families were employed and amplified by polymerase chain reaction and then quantitated by HPLC. A heterogeneous pattern of V beta usage was seen in the fresh TIL; however, V beta 2, V beta 3, V beta 6, V beta 7, V beta 8, and V beta 13.1 were found in increased proportions in at least three of five patients. In the 7-wk tumor-specific TIL, V beta analysis showed an increased usage of V beta 2, V beta 3, V beta 6, and V beta 7 in more than three of five patients. No significant change in V beta representation was seen in control populations that were not stimulated with tumor. Looking at the percent change in V beta usage between fresh and 7-wk tumor-specific cultures, V beta 2 and V beta 6 were augmented significantly in at least three of five patients (108% and 61%, respectively). To verify that the increase in representation of these V beta families was responsible for the increased cytotoxicity observed, mAb specific for V beta 2 and V beta 6 were used to block tumor lysis. Anti-V beta 6 and anti-V beta 2 significantly blocked cytotoxicity against autologous tumor cells in those TIL populations expressing increased levels of these V beta families. These data suggest that a selective repertoire of TCR V beta genes is used to recognize the Ag-HLA class 1 complexes on the surface of ovarian tumor cells, and specifically V beta 2 and V beta 6 appear to mediate antitumor activity. These findings may aid in the development of a more specific immunotherapy in ovarian cancer.

HLA-A2 presents shared tumor-associated antigens derived from endogenous proteins in ovarian cancer.

Tumor-associated lymphocytes (TAL) from the malignant ascites and tumor-infiltrating lymphocytes (TIL) from the solid tumor were isolated from six consecutive untreated ovarian cancer patients. Tumor-specific CTL were generated from both TAL and TIL using solid phase anti-CD3, low dose IL-2 (50 IU/ml), and repeated tumor stimulation. The specificity of TAL and TIL was tested in standard cytotoxicity assays using autologous tumor, several allogeneic ovarian tumors, and the NK-sensitive cell line, K562. Anti-HLA-A-B-C mAb, W6/32, was used to demonstrate that these tumor-specific TAL and TIL were HLA class I-restricted. The ability of the ascitic and solid tumor to present Ag by HLA class I was assessed using Brefeldin A, a fungal metabolite that blocks the endogenous Ag-processing pathway in the viral model. Brefeldin A significantly inhibited tumor-specific cytotoxicity as well as HLA class I expression on the cell surface, suggesting an endogenous source of tumor-associated Ag. Despite previous reports of antigenic heterogeneity in ovarian cancer, shared tumor-associated Ag were shown to exist in this disease as demonstrated by significant allogeneic recognition of HLA-A2-matched patients as opposed to unmatched controls. Specifically, CTL from HLA-A2+ patients lysed HLA-A2+ allogeneic targets significantly better than HLA-A2- allogeneic or HLA-A2+ melanoma targets. There was no such difference with HLA-A2- effectors. Furthermore, HLA-A2 was confirmed to be a major restriction element in ovarian cancer by the blocking of HLA-A2+ effectors against both autologous and allogeneic HLA-A2+ targets with the anti-HLA-A2 mAb, BB7.2. These findings verify a similar lymphocyte/tumor interaction as has been documented in melanoma, suggesting a common mechanism of recognition of these human tumors by lymphocytes.

The influence of angiogenesis inhibitor AGM-1470 on immune system status and tumor growth in vitro.

The synthetic angiogenesis modulator-1470 O-(chloroacetyl-carbamoyl, AGM-1470) is a potent inhibitor of neovascularization. We have investigated the potential influence of this inhibitor in tumor immunobiology using both in vivo and in vitro models. Mice given a single tail-vein injection of tumor cells were later treated wtih AGM-1470 by s.c. injection. After tumor injection, the lungs were evaluated for macroscopic tumor nodules. AGM-1470 significantly reduced the development of macroscopic pulmonary disease but did not eliminate disease. However, tumor-bearing mice treated with AGM-1470 had significantly reduced spleen weight compared to controls. To determine if the observed decrease in spleen weight in the treated animals was associated with immunosuppression, we studied the possible immunomodulatory effects of AGM-1470. AGM-1470 induced no changes in spleen-cell viability compared to controls. However, addition of angioinhibin at the beginning of IL-2-induced spleen-cell activation significantly inhibited the development of NK-mediated tumor-cell killing. Similarly, splenic T-cell proliferation induced by a mitogenic monoclonal antibody to murine T cells was significantly inhibited when activated in the presence of AGM-1470. The in vitro studies were extended by evaluation of immune system status in tumor-bearing mice treated with AGM-1470. In vivo therapy with AGM-1470 did not significantly change the mean splenic lymphocyte counts and CD4/CD8 ratios from control values. In addition, the induction of splenic NK-mediated tumor killing with IL-2 as well as mitogen-induced T-cell activation was not significantly different from control values. These results suggest that AGM-1470 inhibits tumor growth by blocking neovascularization and may, under certain conditions of drug administration, inhibit immune system function.

CD4+ T cell clones isolated from human renal cell carcinoma possess the functional characteristics of Th2 helper cells.

The underlying cause for the observed poor antitumor activity of lymphocytes resident, in situ, in freshly resected human renal cell carcinoma (RCC) is unknown. To examine the basis for this observation we evaluated 217 T cell clones established from 20 consecutive patients with renal cell carcinoma. Of these, 75% were CD4+, 22% were CD8+, and 3% were NK+. Cytotoxic T cell function of CD4+ and CD8+ T cell clones was assessed by antibody-induced triggering of the TcR/CD3 complex. Nearly 93% of the CD4+ clones possessed poor cytolytic activity defined as < 50% killing (E:T, 50:1). Alternatively, nearly 80% of CD8+ clones possessed strong cytolytic activity defined as > or = 50% killing. In all T cell clones tested, MHC nonrestricted killing against Daudi or K562 was not observed. Overall, 77% of the T cell clones isolated from patients with RCC in this study were characterized by poor antitumor cytotoxic function; only 22% of the clones displayed strong cytolytic activity. Clones with strong cytotoxic function were tested for cytotoxic function against autologous tumor. However, all clones tested demonstrated little to no tumoricidal activity against autologous tumor. These results indicate that cytolytic T cells, while present in RCC, are not effectively activated by tumor to express CTL function. The immunobiology of CD4+ T cell clones was further evaluated. Thirty-two CD4+ clones from seven patients revealed distinct patterns of cytokine production following TcR/CD3 activation. CD4+ clones could be divided into IL-2 nonproducers and IL-2 producers. A total of 27/32 clones did not produce IL-2 following activation and IFN-gamma production by IL-2 nonproducers was more than threefold less than that by IL-2 producers. However, no differences were found in the levels of IL-4, IL-6, GM-CSF, or TNF-alpha between the two groups. Antitumor cytotoxicity mediated by CD4+ clones did not correlated with cytokine production. These results demonstrate that among T cells resident in human RCC, the predominant type of lymphocyte population consists of noncytolytic helper CD4+ T cells capable of secreting IL-4, but importantly not IL-2, and low levels of IFN-gamma following activation, thus resembling murine Th2 cells. Only a minor contribution by Th0-like cells was observed (i.e., CD4+ clones capable of secreting IL-2, IL-4, and IFN-gamma following activation).(ABSTRACT TRUNCATED AT 400 WORDS)

T-cell recognition of ovarian cancer.

BACKGROUND: The existence of a tumor-specific T-cell immune response to human malignant melanoma has been well documented. In contrast, the existence of tumor-specific cytotoxic T lymphocyte to ovarian cancer remains controversial despite the abundant lymphocytic infiltrates in the malignant ascites and solid tumor of these patients. METHODS: Tumor-associated lymphocytes (TAL) from the malignant ascites and tumor-infiltrating lymphocytes (TIL) from the solid tumors were isolated from six untreated patients with ovarian cancer. TAL and TIL were grown with initial anti-cluster of differentiation of T cells (CD3), low-dose interleukin-2, and tumor stimulation. T-cell lines were analyzed in functional studies. RESULTS: At 5 weeks, TAL and TIL from five of six patients were > 50% CD8+, and one of six was > 70% CD4+. In all five pairs of CD8 positive cultures, both TAL and TIL exhibited high levels of tumor-specific cytotoxicity for ascite and solid tumor, respectively. T-cell recognition of tumor was mediated through the T-cell receptor-CD3 complex and was human leukocyte antigen class I restricted. TAL and TIL lysed autologous ascitic tumor equally well; however, TAL-mediated tumoricidal activity against autologous solid tumor was consistently and significantly poorer than TIL-mediated killing. CONCLUSIONS: Tumor-specific cytotoxic T lymphocytes can be expanded from both TAL and TIL. However, TAL do not kill solid tumor as efficiently as TIL. This suggests the requirement of TIL, or a combination of TIL and TAL, for effective immunotherapy.

Immunomodulatory effects of interleukin-12 on human tumor-infiltrating lymphocytes.

Interleukin (IL)-12 is a recently described cytokine with multiple immunoregulatory effects on various lymphoid populations, including peripheral blood lymphocytes (PBL), natural killer (NK) cells, and T-cell subsets. We examined the effects of recombinant IL-12 on the immune function of human tumor infiltrating lymphocytes (TIL) from several different tumor types. At an optimal concentration of 50 IU/ml, IL-12 acted as a co-stimulant for TIL recently activated with anti-CD3 antibodies, giving stimulation indices between 2.6 and 9.9. This co-stimulatory effect was independent of IL-2 and was not inhibited by neutralizing antibody to human IL-2. When IL-2 and IL-12 were used synchronously as co-mitogenic stimuli in culture with activated TIL, the effect on proliferation was additive. This additive effect held true over a range of suboptimal concentrations of IL-2 and in different TIL populations. IL-12 did not enhance non-major histocompatibility complex (MHC)-restricted cytotoxicity in the TIL populations tested. TIL cultured with IL-12 killed more autologous tumor targets than TIL maintained in media alone at identical effector/target (E/T) ratios. This effect on cytotoxicity was not as great as that seen in TIL maintained in low-dose IL-2. Co-culture with IL-12 did not change the phenotype of TIL used in these assays. The importance of these findings and possible uses in immunotherapy are discussed.

Survival characteristics of metastatic renal cell carcinoma patients treated with lymphokine-activated killer cells plus interleukin-2.

The immunologic manipulation of patients with metastatic renal cell carcinoma using lymphokine-activated killer (LAK) cells in conjunction with systemic interleukin-2 (IL-2) has been examined under conditions in which the life-threatening toxicities associated with IL-2 treatment have been virtually eliminated. We have examined tumor regression in vivo as well as the survival characteristics of 12 patients with metastatic renal cell carcinoma following immunotherapy. Five of 12 (42%) patients experienced tumor regression exceeding 50 percent following treatment. To determine if immunotherapy had influenced the length of survival, all patients were followed until the time of death. Previous studies have characterized the length of survival of metastatic renal cell cancer patients according to a combination of risk factors unique for each patient. In this model, patients were categorized into risk groups based on the number of risk factors. Survival was found to be dependent on risk factors such as performance status, time from initial diagnosis, number of metastatic sites, recent weight loss, and prior cytotoxic chemotherapy. On completion of the LAK cell immunotherapy protocol, patients were categorized as nonresponders or responders. In addition, they were assigned to risk groups based on their unique profile of risk factors at the time of entry into the protocol. Using this model, we found the median survival of nonresponders (23 months) to be no different from responders (24 months), p > 0.05. This was directly attributable to differences in risk factors which characterized members in these two response groups. However, the observed median survival of nonresponders following therapy was 1.9-fold longer than their projected survival based on the risk factors. Furthermore, the observed median survival of responders was 3.4-fold longer than projected from their risk factors. These results suggest that regardless of response status to therapy, cellular immunotherapy may play a role in mediating a significant palliative effect on the metabolic characteristics of these patients leading to extended survival.

Lymphokine activated killer cells enhance IL-2 prevention of sepsis-related death in a murine model of thermal injury.

It has previously been shown by this laboratory that immunomodulation of thermally injured animals with low-dose interleukin-1 (IL-2) and indomethacin (Indo) improves survival following septic challenge. Lymphokine-activated killer (LAK) cells have been shown to be effective in certain viral infections and to act in synergy with IL-2 in the treatment of certain types of cancer. We have studied the effect of LAK cells in combination with IL-2 and Indo in a murine model of thermal injury and sepsis. Male A/J mice received a 25% scald burn injury or sham burn and were randomized into five groups: (a) sham/vehicle, (b) burn/vehicle, (c) burn/IL-2 (250 U) + Indo (5 micrograms), (d) burn/LAK cells (2 x 10(6) cells), or (e) burn/LAK cells+IL-2+Indo and were treated accordingly for 6 days following injury. LAK cells were generated by in vitro IL-2 treatment of syngeneic spleen cells for 72 hr and cytotoxic activity was confirmed by standard 51Cr release assay using natural killer (NK)-sensitive and NK-resistant targets. In the groups receiving LAK cells they were administered on Day 1 and Day 6 postinjury. On Day 10, septic challenge by cecal ligation and puncture (CLP) or splenectomy, for in vitro studies, was performed. Five-day survival after CLP was 80% in the sham/vehicle group compared to 0% in the burn/vehicle group (P < 0.01). IL-2/Indo and LAK/IL-2/Indo improved survival to 25% (P < 0.05) and 57.1% (P < 0.01), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Secondary cytokine production by lymphoid cells used in cellular immunotherapy.

Interleukin-2 (IL-2) has been used extensively in cellular immunotherapy trials as a systemic activator of the immune system as well as an ex vivo stimulant for lymphoid cell function. Despite the measurement of several in vitro and in vivo immunologic parameters related to cellular immunotherapy, determinants of successful cellular immunotherapy remain unknown. To further delineate the consequences of exposing peripheral blood lymphocytes to high concentrations of IL-2, we assessed the supernatants of IL-2-activated peripheral blood lymphocytes for production of tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma). Exposure of normal monocyte-depleted peripheral blood mononuclear cells (PBMC) to IL-2 caused a dose-dependent increase in secretion of TNF and IFN-gamma which increased linearly after 48 h in culture. Analysis of positively selected, highly purified PBMC subpopulations exposed to IL-2 revealed that TNF-alpha and TNF-beta were produced by both CD3+ and CD16+ subpopulations but not by CD22+ cells. These studies were extended to supernatants obtained from PBMC cultures used in the adoptive cellular immunotherapy of patients with advanced cancer. Patients treated with lymphokine-activated killer (LAK) cell immunotherapy were classified as responders (N = 14) or non-responders (N = 17) to therapy. We found no significant difference in the production of TNF between responders and nonresponders (22 +/- 9 U ml-1 vs. 20 +/- 6 U ml-1), P > 0.05. However, LAK cell supernatants harvested from non-responders contained a significantly higher level of IFN-gamma (232 +/- 94 U ml-1) compared with responders (42 +/- 14), P < 0.05. Furthermore, the linear association between IFN-gamma and TNF-alpha production was different between these two response groups (rs = -0.19 for non-responders and rs = 0.48 for responders). These results suggest that secondary cytokine production by adoptively transferred lymphocytesmay play an important role in the host response to cellular immunotherapy.

The biological effects of immunosuppression on cellular immunotherapy.

Cytoreductive chemotherapy and immunosuppression have been postulated as possible adjuncts to cancer immunotherapy in studies using murine tumour-infiltrating lymphocytes (TIL). Treatment of animals with cyclophosphamide (Cy) therapy alone caused two distinct biological activities that altered the relationship between host and tumour. These two in vivo activities were distinguished by altering the timing and dose of Cy administration relative to tumour implantation. Cy administered 3 days following tumour injection caused a significant decline in the number of pulmonary micrometastases and greater survival compared to untreated controls in proportion to the dose of Cy administered. Further reduction in pulmonary disease was observed when Cy-treated mice were given TIL therapy. The possible role(s) of Cy-induced immunosuppression was studied by injecting Cy 24 h prior to tumour injection. This treatment failed to cause the cytoreductive effect observed when Cy was administered 3 days after tumour since Cy-administration prior to tumour resulted in a significantly higher number of pulmonary metastases and diminished survival compared to untreated controls. Despite the increased number of pulmonary metastases and decreased survival in mice treated with Cy before administration of tumour, therapy with TIL significantly diminished pulmonary disease compared to animals treated with Cy alone. Immunosuppression (without concomitant cytoreductive therapy) prior to TIL treatment significantly prolonged survival. Additional studies with TIL therapy indicate that the survival of animals immunosuppressed prior to tumour injection was significantly longer than controls which received immunotherapy alone. These results suggest that the combustion of immunosuppression plus cellular immunotherapy, which leads to significant survival advantage in these murine tumour models, may possibly augment the clinical response in human TIL trials.

The systemic complement activation caused by interleukin-2/lymphokine-activated killer-cell therapy of cancer causes minimal systemic neutrophil activation.

Twenty-three cancer patients undergoing therapy with interleukin-2 and lymphokine-activated killer cells were studied for evidence of complement activation and systemic neutrophil activation occurring during the course of therapy. Patient plasma samples demonstrated evidence of marked complement activation, with 3-fold elevations of C3a desArg concentrations by the 8th day of therapy. Concentrations of C4a desArg were also elevated by the end of therapy. In vitro chemotaxis of patients' neutrophils both to C5a and to the synthetic peptide chemotaxin, FMLP, was initially normal and then fell progressively to 60% of normal by the end of treatment. Mean neutrophil cell-surface expression of complement receptor Type 1 and complement receptor Type 3 increased in inverse temporal relationship to the deficit in chemotaxis, but showed no consistent pattern for individuals and was only doubled at maximum. Thus, despite a degree of complement activation which should have produced pronounced neutrophil activation, the response of the circulating neutrophils was diminished. In view of this discrepancy, the toxicity of this therapy may not be mediated by activation of circulating neutrophils.

Adaptation of the Müller method to allow quantitative characterization of the affinity and cross-reactivity of antibodies by competitive radioimmunoassay.

A quantitative expression is derived for the evaluation of antigen-antibody affinity constants from radioimmunoassays for the completely general situation in which antigen and antibody are both multivalent. The theoretical analysis is then extended to encompass quantitative characterization of the competitive inhibition observed in screening tests for cross-reactivity of antibody with structural analogs of the eliciting antigen. These procedures are illustrated with a radioimmunological study of the cross-reactivity of a desipramine-elicited monoclonal antibody with other tricyclic antidepressants. An unexpected finding to emerge from this immunochemical study is the demonstration that a single affinity constant suffices to describe the interaction of desipramine with a polyclonal antibody elicited by this univalent antigen.

Approaches to immunotherapy of cancer: characterization of lymphokines as second signals for cytotoxic T-cell generation.

Lymphokines, the soluble molecules produced by cells of the immune system, regulate cell-cell interactions and, consequently, the functional status of the immune system. Altering immunoregulatory pathways with lymphokines in vivo may provide a mechanism for controlling a variety of immunologic disorders. Although normally produced in vivo in very small quantities, the widespread availability of recombinant lymphokines has made it possible to study the molecular signals involved in production of lymphocyte effectors with activity against tumor. For example, interleukin-2-based cancer immunotherapy programs have, in certain clinical situations, suggested that immunologic intervention can influence the regression of metastatic cancer. Ultimately the successful application of these biologic agents requires an understanding of the interaction between the immune system and tumor on a molecular level. To induce a given biologic effect, it is necessary both to classify the required lymphokines and to identify the relevant effector cell populations. This review will examine the progress made in identifying the requirements for lymphokine-induced cytotoxic T-lymphocyte function.

Reduction of smooth muscle hyperplasia in vein grafts in athymic rats.

While developing an animal model of a vascular malformation, we found that two doses of cyclosporin A significantly reduced the smooth muscle cell hyperplasia observed in vein-arterial interposition grafts in Sprague-Dawley rats. Therefore, we hypothesized that T cells may either produce or augment a mitogen for vascular smooth muscle cells. To further investigate this, we quantitated the extent of smooth muscle cell hyperplasia in the vein grafts of athymic nude rats that lack mature, functioning T cells. Male Sprague-Dawley rats (275 to 350 gm) and athymic nude rats were anesthetized, and a segment of the superficial epigastric vein was placed into the transected femoral artery using microsurgical techniques. Sprague-Dawley rats (N = 9) and athymic nude rats (N = 5) undergoing vein grafting received 30 mg/kg cyclosporin A intraperitoneally, intraoperatively and 24 hours later. Sprague-Dawley rats (N = 7) and athymic nude rats (N = 6) that had vein grafts were not treated with cyclosporin A. Animals were killed at either 3 weeks or 6 weeks and histologic sections were taken from the middle of the graft to avoid clamp-induced trauma. At 3 weeks, untreated vein grafts in Sprague-Dawley rats exposed to arterial pressure exhibited a nine-fold increase in smooth muscle hyperplasia compared with the preoperative vein. Treatment of Sprague-Dawley rats with cyclosporin A resulted in a 57% reduction of smooth muscle hyperplasia (p less than 0.05). Vein grafts from athymic nude rats exhibited a 51% reduction in smooth muscle hyperplasia (p less than 0.05). Sprague-Dawley rats killed at 6 weeks revealed a recovery of smooth muscle hyperplasia equivalent to an untreated Sprague-Dawley vein graft at 3 weeks. Inhibition of smooth muscle hyperplasia persisted for 6 weeks in the athymic nude rats. Cyclosporin A administration or T cell deficiency in athymic nude rats decreases the smooth muscle hyperplasia observed in venous grafts exposed to arterial pressure. This finding provides evidence for a possible role of T cells in the regulation of cell growth in the vascular wall.

The role of interleukin-2 in cancer immunotherapy.

The arena of cellular immunotherapy is an emerging field of study. The field has strong foundations in numerous animal tumor models, which provide avenues of research for refinements in human immunotherapy. Experience from a number of research centers can be generalized: LAK cell trials in humans have demonstrated that PBMCs activated with IL-2 possess reproducible antitumor activity in vitro. Exogenous IL-2 administration to patients is required to maintain the biologic activity of the activated cells, although the requirement for LAK cells in tumors like melanoma is not clear-cut. Overall, the response rate to immunotherapy in humans is approximately 30%. The toxicity of immunotherapy, which is related to the dose of systemic IL-2, can be significantly reduced. The next generation of trials of cellular immunotherapy in humans will use TILs. Murine experiments indicate that this population of cells is more powerful and potentially more specific for tumor than LAK cells. Like LAK cells, TILs also require exogenous IL-2 for optimum performance in vivo. The difficulty in reproducible TIL growth can be overcome by the use of monoclonal antibody activation and IL-2. Improvements in TIL therapy are anticipated from the use of TILs combined with hybrid antibodies, the addition of other biologic response modifiers, and the implementation of genetically engineered cells and cell products.

Solid-phase anti-CD3 antibody activation of murine tumor-infiltrating lymphocytes.

Seventeen consecutive s.c. murine tumors, derived from a sarcoma and a colon adenocarcinoma, were cultured in the presence of recombinant interleukin 2 (rIL-2) for growth of tumor-infiltrating lymphocytes (TIL). Identical cultures were activated by solid-phase monoclonal antibody directed against the murine CD3 epsilon-chain, in conjunction with rIL-2. Forty-eight h later, cells were replaced in rIL-2 alone. Proliferation of anti-CD3-stimulated cultures was 1- to 17-fold greater than those cultured with rIL-2 alone (P less than 0.05). Both culture conditions yielded TIL which stained greater than 80% Thy-1.2+/Lyt-2+ (P greater than 0.05), less than 7% Thy-1.2+/L3T4+ (P greater than 0.05). Regardless of culture condition, longitudinal studies of in vitro cytotoxicity generated from 10 TIL preparations revealed no significant differences between the ability of TIL to lyse the murine natural killer-sensitive line YAC or heterologous or autologous tumor (P greater than 0.05). In vivo antitumor activity of TIL was tested by the adoptive transfer of suboptimal doses of TIL plus systemic rIL-2 to mice with pulmonary micrometastatic disease. No difference in tumor regression was noted between the TIL cultured with anti-CD3 plus rIL-2 or with rIL-2 alone (P greater than 0.05). Anti-CD3 stimulation of murine TIL cultures significantly increases lymphocyte cell yield without alteration of their phenotype, in vitro tumoricidal activity, or in vivo therapeutic effect.