Divergent effects of 4-1BB antibodies on antitumor immunity and on tumor-reactive T-cell generation.

4-1BB is an inducible receptor-like protein expressed rapidly by both CD4 and CD8 T-cells after activation. 4-1BB cross-linking, either by binding to 4-1BBL or by antibody ligation, delivers a costimulatory signal to enhance T-cell activation and proliferation. Previous studies have demonstrated that the administration of 4-1BB monoclonal antibodies (mAbs) induces antitumor immune responses. In the current study using several murine tumors, we examined the systemic effects of 4-1BB mAb on the growth of s.c., intracranial (i.c.), and pulmonary metastases. In addition, the effects of 4-1BB mAb on the generation of antitumor effector T cells were examined. Treatment of 3-day i.c. MCA 205 sarcoma and GL261 glioma with the antibody resulted in prolongation of survival and cure of disease in some mice, whereas only minimal therapeutic effects were observed in established s.c. and pulmonary tumors. No antitumor effects against the poorly immunogenic B16/D5 melanoma were observed. Interestingly, successful treatment of i.c. tumors induced concomitant regression of s.c. tumors. Experiments using severe combined immunodeficient mice and mice depleted of either CD4 or CD8 T cells demonstrated T-cell dependence of the antitumor effects. For generation of effector T cells in the tumor-draining lymph nodes (LNs), administration of 4-1BB mAb had adverse effects, despite the apparent hypertrophy of the LNs. During in vitro activation of tumor-draining LN T cells with anti-CD3 and interleukin 2, the 4-1BB mAb augmented proliferation, resulting in an increase in CD8 T cells. However, they were less therapeutic than not treated LN cells. In adoptive immunotherapy, the coadministration of 4-1BB mAb enhanced the therapeutic efficacy. These results thus demonstrate the limits and potential advantages of 4-1BB antibody interactions with antitumor T cells in vivo and in vitro and suggest that therapeutic interactions of the antibody may be used in a variety of immunotherapeutic approaches.

Coinfusion of irradiated splenocytes with low titer tumor-infiltrating lymphocytes augments antitumor efficacy in adoptive immunotherapy.

We hypothesized that adoptively increasing the density of antigen-presenting cells (APCs) at a tumor site would improve tumor-infiltrating lymphocyte (TIL) in vivo antitumor efficacy. Irradiated splenocytes were used as crude APCs. Alone, they did not have in vitro antitumor activity nor did they augment TIL efficacy in vitro. Pulmonary metastases were established by intravenous (i.v.) injection of 5 x 10(5) MC-38 tumor into irradiated C57B1/6 mice (500 cGy). After 3 days, MC-38 TIL (0.1, 0.5, and 1 x 10(6) cells) +/- irradiated splenocytes (5,000 cGy) as APCs were administered intravenously (0.25, 0.5, and 1 x 10(6) cells) to each group (n = 5/group). Interleukin-2 (60,000 IU) was injected intraperitoneally three times daily for 3 days. Mice were sacrificed 9 days later and metastases elaborated in blinded fashion. A titer of 1 x 10(6) TIL, completely eradicated pulmonary metastases. In two consecutive experiments, when increasing titers of irradiated splenocytes were coinfused with a constant titer of TIL that did not completely eradicate pulmonary metastases, a moderate reduction in pulmonary metastases was observed. The contribution of splenocytes to an improvement in TIL antitumor efficacy was not altered when irradiated splenocytes derived from mice bearing 10-day subcutaneous MC-38 tumors were used. The coinfusion of nonirradiated splenocytes did not improve TIL antitumor in vivo activity. Activated B cells (expressing ICAM-1, B7.1, and B7.2) had no effect on in vitro tumor lysis and did not augment in vivo TIL efficacy. The results show a modest but statistically significant improvement in adoptive immunotherapy antitumor efficacy with fewer TIL by coinfusion of irradiated splenocytes. Further studies to characterize the active potential APC cell subpopulation and to clarify the mechanism(s) responsible for in vivo augmentation of TIL antitumor efficacy are in progress.

A long-term analysis of 620 patients with malignant melanoma at a major referral center.

BACKGROUND: A univariate and multivariate statistical analysis of a single surgeon's experience with resectable malignant melanoma during 26 years (November 1970 to August 1996) was conducted. METHODS: Six hundred twenty consecutive patients were registered. Univariate analysis of disease-free survival (DFS) and melanoma survival (MS) was calculated by the Kaplan-Meier method and correlated to American Joint Committee on Cancer stage, thickness, ulceration, site, lymph node involvement, age, sex, type, and excision margins. Linear trends, log-rank test, and pairwise comparisons were used to discriminate differences in survival curves. A Cox proportional hazards model was used for multivariate analysis and determination of relative risk. RESULTS: Univariate analysis of stage, thickness (in millimeters), ulceration, lymph node involvement, age, type, and margins of excision were predictive of DFS (5 years, 85.7%; 10 years, 82.5%) and MS (5 years, 92.2%; 10 years, 87.8%) (P < .01). Multivariate analysis revealed correlations with thickness, ulceration, and age in predicting DFS (relative risk = 2.75, 2.21, and 1.47, respectively) and MS (relative risk = 2.66, 2.47, and 1.48, respectively). The 5-year MS rate was 73.3% and 93.3% for patients with positive and negative lymph nodes, respectively. Of 133 patients who underwent lymph node dissection, 28 (21.1%) had nodal metastases. Patients with primary melanomas thicker than 4 mm had 50% metastatic involvement of their lymph nodes. CONCLUSIONS: Our findings reveal that thickness, ulceration, and age are the most important predicting factors in DFS and MS. The data support including ulceration and age in modifying American Joint Committee on Cancer staging for melanoma.

RANTES secretion by gene-modified tumor cells results in loss of tumorigenicity in vivo: role of immune cell subpopulations.

An immunogenic murine fibrosarcoma cell line was genetically modified to express and produce the human RANTES chemokine stably. In in vitro chemotaxis assays purified recombinant human RANTES as well as human RANTES secreted by the modified murine tumor cells were strongly chemoattractant for mouse CD8+ /Thy-1+ tumor-infiltrating lymphocytes (TIL). RANTES production did not alter the growth of these cytokine gene-modified tumor cells in vitro, but injection of RANTES-secreting cells resulted in the abolition of the ability of those cells to form solid tumors in vivo. The growth of tumors could be restored by co-administration of monoclonal antibodies that inhibit the function of various subsets of immune cells. For example, depletion of CD8+ T cells by antibody administration resulted in complete restoration of solid tumor formation by RANTES-secreting cells, whereas depletion of the CD4+ T cell population resulted in a partial restoration of tumor formation. Additionally, administration of an anti-CR3 monoclonal antibody known to inhibit the in vivo migration of macrophages also completely restored the tumorigenicity of RANTES-secreting fibrosarcoma cells. Thus, the human RANTES chemokine can abolish tumorigenicity of an immunogenic fibrosarcoma in an in vivo murine model, and this process is mediated by various subpopulations of immune effector cells.

A tumor-elaborated supernatant factor chemotactic for IL-2 expanded tumor infiltrating T-lymphocytes.

Very little is known about factors influencing the migration of highly activated T-lymphocytes. One such lymphocyte population is the IL-2 expanded population of T cells infiltrating tumors. These tumor-infiltrating lymphocytes (TIL) can cause tumor regression in patients with metastatic cancer and in murine tumor models when given in adoptive transfer. In patients with melanoma, these TIL have been shown to migrate to sites of tumor and this may be a critical factor in their antitumor activity. In this study, a 48-well microchemotaxis chamber and a 5 microns pore nitrocellulose filter membrane system was utilized to study the motility of murine TIL. A chemotactic response was observed to supernatants from freshly explanted, autologous, and nonautologous tumor cultured for 24 h. Serially passaged autologous and nonautologous tumors also produced supernatants with chemotactic activity. Supernatants from single cell suspensions of normal tissues prepared and cultured identically did not elicit chemotaxis. Chemotactic activity for TIL was not removed by dialysis (2000 MW exclusion limit), its activity was undiminished by heat treatment at 60 degrees C for up to 60 min, and it was trypsin sensitive. Tumor supernatants were also chemotactic for two IL-2-dependent specifically alloreactive CTL lines (CTL-TIM and OE-4), but not two helper T cell lines (D-10 and D-1.5) or normal resting lymphocytes. This is the first demonstration of a chemotactic effect on IL-2-dependent, activated T cells. Characterization and purification of factors from tumor responsible for this directed migration are in progress.

Bilateral aortoiliac pressure measurements from a single puncture site.

Bilateral aortoiliac pressure measurements were obtained at the time of aortography by performing contralateral iliac artery catheterization in 102 patients with peripheral vascular disease. Patients studied had mild-to-moderate aortoiliac disease and in their cases the additional information was believed to be important in therapeutic decision making. Patients with severe ulcerative disease, peripheral thromboembolism, and significant aortic aneurysms were excluded. Pressure gradients out of proportion to the degree of stenosis as measured on the arteriograms were frequently found, particularly during high flow states with intraarterial injections of a vasodilator. There were two failures of catheterization and three minor complications, none embolic. We conclude that contralateral iliac artery catheterization in selected patients is both safe and generally easily performed by experienced angiographers.

Pharmacokinetics and tissue distribution of parenterally administered human alpha-lymphotoxin in normal and meth-A tumor-bearing BALB/c mice.

These in vivo studies examine the pharmacokinetics of parenterally administered purified, native human alpha-lymphotoxin (LT) in normal and Meth-A bearing BALB/c mice. We found that the lytic activity of alpha-LT was inactivated within 5 h in the blood of both normal and tumor-bearing mice in vivo. However, LT bioactivity in vitro was not affected by incubation with fresh serum. Radioiodinated LT was rapidly sequestered in the kidneys of both normal and tumor-bearing animals. Systemically administered, radioiodinated LT did not selectively localize in tumor tissues.

Differences in the bioactivity of recombinant human TNF, LT, and T-cell-derived LT-3 on transformed cells in vitro and the Meth A tumor growing in BALB/c mice.

Cytolytic human T cells and the continuous human T cell line YM 1.2 can secrete a lymphotoxin (LT) form, termed LT-3, when stimulated in vitro. LT-3 is immunologically related to, but functionally distinct from, both alpha LT and tumor necrosis factor (TNF). Purified, native LT-3 and recombinant human LT and TNF were tested for their ability to destroy a panel of primary and continuous cell lines in vitro and cause necrosis of 8- to 9-day cutaneous Meth A tumors growing in BALB/c mice. LT-3 is more effective than either LT or TNF in inducing destruction of continuous cell lines in vitro. LT-3 induces destruction of cell lines that are resistant to both TNF and LT, and much less LT-3 is required to destroy cells that are LT and/or TNF sensitive. In contrast, none of these mediators had any measurable negative effects when tested on primary human fibroblasts. Additional studies revealed that LT-3 is also active in vivo and can cause necrosis of cutaneous Meth A tumors when administered intraperitoneally or intratumorally on BALB/c mice. LT-3 induces more rapid, consistent, and complete tumor necrosis than equivalent doses of either LT or TNF.

Purified native and recombinant human alpha lymphotoxin [tumor necrosis factor (TNF)-beta] induces inflammatory reactions in normal skin.

These studies report findings that demonstrate that human alpha lymphotoxin (LT) induces local, visible, and microscopic inflammatory reactions in normal skin. Skin sites in rabbits, when inoculated with a single injection of native or recombinant human alpha lymphotoxin, demonstrated erythema, swelling, and warmth within 5 hr. Erythema peaked between 24 and 48 hr had resolved by 72 hr. Histologic studies of skin sites injected with native LT revealed polymorphonuclear neutrophil (PMN) infiltration and edema beginning as early as 3 hr posttreatment. Individual skin sites that received three daily injections of native LT exhibited persistent erythema and swelling. Palpable induration was evident 24 hr after the second injection in the series. Histologic examination revealed the presence of many PMNs with associated focal dermal destruction, in the form of microabscesses, and scattered mononuclear cells. In contrast, control materials and recombinant human tumor necrosis factor (TNF-alpha) did not induce visible skin reactions in the rabbit. Several additional controls excluded endotoxin as being the agent responsible for the inflammatory skin reactions observed. The ability of LT to induce inflammation may have a role in its antitumor activity and it may be an important endogenous mediator in other immunologic reactions.

Human alpha lymphotoxin (LT): studies examining the mechanism(s) of LT-induced inflammation and tumor destruction in vivo.

Recent information from our laboratories indicates that human alpha lymphotoxin (LT) is a powerful mediator of tumor necrosis, and inflammation. Our studies indicate that LT induced effects in vivo are complex and involve multiple mechanisms(s). The data presented here suggests that LT induced tissue destructive and inflammatory reactions mediated by cytolytic and antigen reactive lymphocytes may have a role in a much broader variety of inflammatory processes than had been appreciated.

Electrophysiologic changes associated with chronic administration of organophosphates.

Changes in the electrophysiologic activity of the rat sciatic nerve were examined after repeated dosing with either of two organophosphate insecticides, parathion, and trichlorfon. Although the parathion treated animals showed overt signs of systemic toxicity, there were no significant changes in any of the measured parameters of sciatic nerve excitability. Trichlorfon, on the other hand, produced dose-dependent changes in the duration, rise time, relative area, and refractory period of the sciatic nerve compound action potential. The observed changes indicated an increased excitability of the nerve. During the early development of these electrophysiologic changes there were no accompanying histologic changes in the nerve. This suggests that changes in nerve excitability may be a sensitive indicator of neurotoxicity, and that continued trichlorfon exposure may lead to a cumulative alteration in nerve function.