Complexes of platelet factor 4 and heparin activate Toll-like receptor 4.

BACKGROUND: In some patients, the anticoagulant heparin elicits formation of antibodies that can cause the life and/or limb-threatening syndrome known as heparin-induced thrombocytopenia (HIT). HIT antibodies target complexes formed at specific molar ratios of heparin and platelet factor 4 (PF4). The unpredictable occurrence and the mechanism of this atypical immune response to PF4:heparin complexes are poorly understood. OBJECTIVE: We investigated whether complexes formed at specific PF4:heparin ratios (PHRs) might resemble molecular patterns associated with host defense responses. METHODS: We used an in vitro cytokine release assay to determine whether defined PHRs caused cytokine release from human whole blood. Lipopolysaccharide (LPS) was used as a positive assay control, and some experiments included antibodies to block Toll-like receptor 4 (TLR4). RESULTS: PF4:heparin complexes caused release of the biomarker interleukin 8 in whole blood, and the level of response varied with the stoichiometric ratio of PF4 to heparin. The profile of response to LPS and to PF4:heparin complexes varied among blood donors, and the interleukin 8 response to both LPS and PF4:heparin was inhibited by TLR4-blocking antibodies. CONCLUSIONS: Specific PF4-heparin complexes can elicit a TLR4-mediated response, suggesting that these complexes can mimic a pathogen-associated molecular pattern, and supporting the suggestion that the HIT immune response represents a misdirected host defense mechanism.

Sustained release of tissue factor following thrombosis of lower limb trauma.

This study was undertaken to provide evidence for the mechanism of venous thromboembolism (VTE) in healthy patients with minor lower limb injury (fracture; Achilles tendon rupture) that was medically managed with plaster cast/brace immobilization. The Plaster Cast clinical trial provided a unique opportunity to identify the natural history of VTE using placebo-controlled patients (n = 183) with validation of the mechanism using the low-molecular-weight heparin (LMWH; reviparin)-treated patients (n = 182). Confirmed VTE in this population was associated with a burst of tissue factor release (and a minor fibrinolytic deficit) leading to thrombin generation that was sustained at least 5 weeks, greater with fractures than with soft-tissue injuries and greater with surgery than with conservative treatment. The root cause likely involves platelet/leukocyte activation (inflammation) rather than endothelial cell injury. Thromboprophylaxis with a low dose of LMWH reduced thrombin generation, with patients undergoing surgery benefitting the most.

Emphasis on the Role of PF4 in the Incidence, Pathophysiology and Treatment of Heparin Induced Thrombocytopenia.

Heparin Induced Thrombocytopenia (HIT) is caused by antibodies that recognize platelet factor 4 (PF4) associated with polyanionic glycosaminoglycan drugs or displayed on vascular cell membranes. These antibodies are elicited by multimolecular complexes that can occur when heparin is administered in clinical settings associated with abundant PF4. Heparin binding alters native PF4 and elicits immune recognition and response. While the presence of heparin is integral to immunogenesis, the HIT antibody binding site is within PF4. Thus HIT antibodies develop and function to cause thrombocytopenia and/or thrombosis only in the presence of PF4. Future emphasis on understanding the biology, turnover and regulation of PF4 may lead to insights into the prevention and treatment of HIT.

A colorimetric, metabolic dye reduction assay detects highly activated platelets: application in the diagnosis of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is caused by antibodies, elicited in response to heparin anticoagulant therapy, which can cause extreme platelet activation and result in a highly procoagulant state. Most diagnostic tests for HIT antibodies measure in vitro platelet activation by detecting aggregation or granule release responses. This study demonstrates that the high level of activation by HIT antibodies leads to a rapid breakdown of platelet metabolic activity. Resting or mildly activated platelets metabolize tetrazolium-based indicator dye to a dark colored product, in proportion to cell concentration and dye incubation time. Highly activated, procoagulant platelets resulting from incubation with HIT antibodies fail to metabolize dye and remain light in color. The loss of ability to metabolically reduce indicator dye provides a colorimetric endpoint that can be used in an in vitro washed platelet activation assay to detect HIT antibodies. In a prospective evaluation, 145 diagnostic specimens were tested concurrently by both the colorimetric, dye reduction assay and the clinical laboratory standard, radiolabeled-serotonin release assay ((14)C-SRA). Results were in agreement for 96-100% of cases, depending on the chosen stringency of assay cut-off values. This study demonstrates that the metabolic dye reduction assay is comparable to the (14)C-SRA for HIT diagnosis. In addition, this novel assay may have even wider applicability, facilitating studies on the physiologic and clinical relevance of highly activated platelet populations.

Physiological changes in membrane-expressed platelet factor 4: implications in heparin-induced thrombocytopenia.

BACKGROUND: Many heparin-induced thrombocytopenia (HIT) antibodies cause platelet activation in the serotonin release assay (SRA) in the absence of heparin. This in vitro observation may help unravel the mechanism of delayed-onset HIT, where seropositive patients develop thrombocytopenia and associated thrombosis after cessation of heparin. OBJECTIVE: Studies were conducted to examine the relationship between platelet environment, surface PF4 expression, and the extent of heparin-independent platelet activation in the SRA. METHODS: Ex vivo platelets were washed and labeled for SRA, then used either before or after 45 minutes of recovery at 37 degrees C. HIT antibody-mediated serotonin release in the absence of heparin was compared to the extent of surface staining of the platelets with fluorescent anti-human PF4 antibodies. RESULTS: Handling of platelets for in vitro studies resulted in transient expression of surface PF4, and it was during this interval that platelets were most sensitive to activation by HIT antibodies in the absence of heparin. Heparin-independent platelet activation was attenuated when SRA-positive specimens were retested after platelets were incubated 45 minutes at 37 degrees C. Surface PF4 expression was diminished on the rested platelets, compared to the same platelets labeled immediately after handling. Thus compared to rested platelets, mildly activated platelets had elevated surface PF4 expression and a higher level of HIT antibody-mediated, heparin-independent platelet activation. CONCLUSION: Surface expression of PF4 reflects HIT antigen presentation, and varies with the physiological state of platelets. Thus there can be differences in HIT antibody target availability among patients which may explain the variability in consequences of HIT antibody seropositivity.

Effect of anti-platelet factor-4/heparin antibody induction on early saphenous vein graft occlusion after coronary artery bypass surgery.

BACKGROUND: Antibodies to complexes of heparin and platelet factor 4 (PF4) are capable of causing heparin-induced thrombocytopenia (HIT). Recent evidence suggests that anti-PF4/heparin antibodies may be prothrombogenic even in the absence of thrombocytopenia and clinically-recognized HIT. OBJECTIVES: To determine if induction of anti-PF4/heparin antibodies is an independent risk factor for early saphenous vein graft (SVG) occlusion or adverse clinical outcome after coronary artery bypass graft (CABG) surgery. PATIENTS/METHODS: Anti-PF4/heparin antibody titers were measured in 368 patients prior to and then 4 days, 6 weeks and 6 months after CABG surgery. Serotonin release assay (SRA) and antibody isotype analysis were also performed on 6-week samples. SVG patency was determined in 297 patients 6 months after surgery by multidetector computed tomography coronary angiography. RESULTS: Six weeks after surgery, 52% of patients were anti-PF4/heparin seropositive and 9% were SRA positive. Six months after surgery, neither the percentage of occluded SVG (19% vs. 20%, P = NS), the percentage of patients with an occluded SVG (33% vs. 33%, P = NS) nor the incidence of adverse clinical events (21% vs. 24%, P = NS) differed between seropositive and seronegative groups. Neither IgG isotype nor SRA positivity was additionally predictive of SVG occlusion or adverse clinical outcome. CONCLUSION: Induction of anti-PF4/heparin antibodies, even those capable of heparin-dependent platelet activation, is not independently associated with early SVG occlusion or adverse clinical outcomes after CABG surgery.

Activation of platelets by heparin-induced thrombocytopenia antibodies in the serotonin release assay is not dependent on the presence of heparin.

The serotonin release assay (SRA) tests for antibodies responsible for heparin-induced thrombocytopenia (HIT). By definition, SRA-positive antibodies cause platelet serotonin release in vitro, in the presence of low concentrations of heparin, but not with excess heparin. Many SRA-positive sera activate platelets in the presence of saline without drug, either as a result of residual heparin in the specimen, or because of intrinsic features of the HIT antibodies. The present experiments show that neither exhaustive heparinase treatment, nor chromatographic removal of heparin abrogates the spontaneous platelet activation caused by these HIT antibodies. This is the first study to systematically demonstrate that in vitro activity of HIT antibodies can be independent of heparin. In addition, T-gel chromatography demonstrated differences among fractions of enzyme-linked-immunosorbent assay (ELISA)-positive HIT antibodies within individual specimens. Certain ELISA-positive fractions had SRA activity while others did not, and the SRA activity was not proportional to HIT antibody ELISA titer. These data suggest that antibodies formed as a result of heparin treatment are heterogeneous, and that some can contribute to the pathogenesis of HIT even when heparin is no longer present.

Incidence of antiplatelet factor 4/heparin antibody induction in patients undergoing percutaneous coronary revascularization.

The incidence of antiplatelet factor-4/heparin antibody formation in patients who receive contemporary doses of unfractionated heparin in the setting of percutaneous coronary revascularization is unknown. Also unknown is the ability of these antibodies to activate platelets or adversely affect clinical outcome in the absence of clinically recognized heparin-induced thrombocytopenia. To address these questions, we serially measured antiplatelet factor-4/heparin antibody levels and performed serotonin release assays in patients who underwent percutaneous coronary intervention. Correlations were then made across antibody induction, heparin exposure, and clinical outcome at 6 months.

Prevalance of heparin-dependent platelet-activating antibodies in preterm newborns after exposure to unfractionated heparin.

Heparin is frequently used in preterm infants to prolong the patency of intravascular catheters. The aim of this study was to evaluate the prevalence of heparin-dependent platelet-activating antibodies in newborns. A cross-section of all preterm newborn infants expected to require heparin to maintain patency of a central venous access line were enrolled. A blood sample was obtained soon after birth before heparin exposure to exclude the possibility of placental transfer of maternal heparin-dependent platelet-activating antibodies. A second sample was obtained at termination of heparin use (mean duration of heparin exposure was 23 +/- 13 days; range, 6-67). Paired samples, at birth and after heparin use, were available for 42 infants with a mean gestational age of 27.8 +/- 2.2 weeks and birth weight of 1036 +/- 267 g. Thrombocytopenia developed in 57% (24/42) of the infants. None of these infants had clinical suspicion of thrombosis during the study period. The etiology of thrombocytopenia was confirmed sepsis in six, presumed sepsis in three, necrotizing enterocolitis in one, and unclear in 14 infants. Anti-heparin/platelet factor 4 antibodies measured using the standard assays for heparin-induced thrombocytopenia (two commercially available enzyme-linked immunosorbent assay tests and the functional platelet serotonin release assay) were negative on all infants. Although it could be related to the poor ability of these infants to mount an immunologic response, further research is necessary to fully understand this lack of response to heparin and to elucidate further the reasons for thrombocytopenia in very-low-birth-weight infants.

Decreased prevalence of heparin-induced thrombocytopenia with low-molecular-weight heparin and related drugs.

Heparin-induced thrombocytopenia (HIT) Type II represents a disease spectrum associated with a high risk of thrombosis leading to limb loss and death. The pathophysiology of HIT is based on the development of antibodies to the heparin-platelet factor 4 (PF4) complex. Unfractionated heparin (UFH) is heterogeneous in molecular chain length and degree of sulfation accounting in part, for, the heterogeneity of HIT antibodies. Because of its smaller size, low-molecular-weight heparin (LMWH) does not interact with PF4 and platelets as efficiently as does UFH. This translates into a lower risk of immune sensitization with LMWH than with UFH treatment. LMWH is less likely than UFH to cause antibody generation and thus patients do not develop clinical HIT at the same frequency with LMWH as with UFH treatment. The antibodies generated by LMWH treatment are more often immunoglobulin A (IgA) and IgM as opposed to IgG antibodies, which are associated with symptomatic clinical HIT generated by exposure to UFH. However, platelet activation/aggregation can occur from LMWHs in the presence of most pre-existing HIT antibodies that had been generated from UFH exposure, although the response is less than that caused by UFH plus HIT antibody. With the expanded use of LMWH, the frequency of clinical HIT may naturally decline, given that LMWHs are less likely to generate HIT antibody.

Newer insights on the mechanism of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) type II is a complex clinical syndrome. It is an immune reaction to heparin in which the formation of antibodies targeted against the heparin-platelet factor 4 complex results in platelet activation. Platelet activation plays a central role in HIT; however, platelet activation does not occur as an isolated physiologic response. To elucidate further the mechanism of thrombogenesis in HIT, we undertook studies to determine the effect of heparin antibodies on endothelial cells, leukocytes, and the inflammatory state. We summarize our previous and new findings. For endothelial cells: Antiheparin antibodies bind to and directly activate microvascular endothelial cells, whereas binding to and activating macrovascular endothelial cells requires preactivation by platelets or tumor necrosis factor alpha (TNFalpha). Increased circulating levels of hemostatic activation factors as observed with thrombosis, particularly soluble P-selectin, plasminogen activator inhibitor type 1 (PAI-1), tissue factor, and thrombomodulin, were associated with endothelial cell activation and were also found in the blood circulation of patients with HIT. For the inflammatory state: Neutrophils and monocytes (but not lymphocytes) bind to and form complexes with platelets in the presence of HIT antibodies. Activated monocytes bind to endothelial cells and produce a procoagulant state. Patients with HIT have an increased level of cytokines in their blood circulation. For HIT antibodies: Only heparin fractions larger than 5 kd interacted with HIT antibodies, explaining why low-molecular-weight heparin (LMWH) usually does not generate antibodies. HIT antibodies are heterogeneous in structure, affinity, and specificity. These data suggest that, in addition to the platelet component, several other mechanisms are associated with the pathophysiology of HIT. These include an inflammatory state, endothelial cell remodeling, and the known procoagulant state. Differences between patients in the levels of the inflammatory markers may relate to various stages of the inflammatory/procoagulant state that exists in patients with HIT. The variations within the HIT antibodies may influence their ability to activate platelets, endothelial cells, and leukocytes, and thus contribute further to the variations in the pathogenicity of HIT.

Heparin-induced thrombocytopenia with associated thrombosis in children after the Fontan operation: report of two cases.

Heparin-induced thrombocytopenia is a widely recognized clinical disorder. The spectrum of disease ranges from clinically insignificant to severe thrombosis (heparin-induced thrombocytopenia with associated thrombosis). Overall, thrombosis occurs in approximately 33% of adults diagnosed with heparin-induced thrombocytopenia and has been associated with high morbidity and mortality rates. Diagnostic testing for this disorder is not standard in children with thrombocytopenia who are receiving heparin, despite the fact that children with congenital heart disease may be exposed to heparin frequently. There are few reported cases of heparin-induced thrombocytopenia with associated thrombosis in children; herein, we describe the cases of 2 children who developed this disorder after undergoing a Fontan operation.

Effects of a heparin-binding protein on blood coagulation and platelet function.

The objective of this study was to characterize the heparin-binding properties of a protein secreted by mouse myeloma cells. The characterization was performed using clinical assays, such as heparin activity assays and heparin-induced thrombocytopenia (HIT) platelet activation assays. The tests were performed in the presence of heparin, low-molecular-weight heparins (LMWH), or heparinoids and either heparin-binding protein (HBP) or saline to determine whether the HBP affects the activity of heparins. The characterization of the HBP using heparin activity assays showed that the HBP shortened the prolonged clotting times of the activated partial thromboplastin time (aPTT) and thrombin clotting time induced by high concentrations of unfractionated heparin. The chromogenic assays for antithrombin (AT), thrombin inhibition, and factor Xa inhibition demonstrated that this effect is related to heparin concentrations below 0.5 IU/ml. The Heptest assay did not detect these differences. The HBP did not modify the anticoagulant effect of any LMWH or low- or high-sulfated glycosaminoglycans in the aPTT assay. Activation of donor platelets in the presence of unfractionated heparin, platelet factor 4 (PF4), and HIT-serum was not counteracted by the HBP in any of the assays. The characterization of the HBP using a PF4-enzyme-linked immunosorbent assay (ELISA) confirmed the lack of structural identity with PF4. However, the optical density data indicated that the protein structure may be similar to PF4 by binding to a PF4 antibody. These data suggest that the HBP isolated from mouse myeloma cells has a low affinity to heparin and interacts with the secondary binding site to AT and also perhaps to PF4.

In vivo indomethacin treatment causes microglial activation in adult mice.

The current study was undertaken to study the role of prostaglandins in regulating microglial activation. Mice were treated with indomethacin (2 microg/ml) in their drinking water to selectively inhibit cyclooxygenase activity. After 4-8 days, the effect of inhibiting prostaglandin synthesis on microglial activity was evaluated. This was accomplished by analyzing microglial expression of Mac-1 (C3 complement receptor) as an indicator of activation. Mac-1 expression was assessed by immunohistochemistry of fixed brain cryosections, and by flow cytometric analysis of immunostained single cell suspensions. Both methods demonstrated that compared to age-matched, untreated controls, brains of indomethacin-treated mice had increased levels of Mac-1 expression, suggesting an increase in the state of microglial activation. These results demonstrate the importance of prostaglandins in down regulating microglial activity, and that inhibition of prostaglandin synthesis with indomethacin may act to increase the reactivity of the brain's immune system.

Pinealocyte ultrastructure in mutant mice that lack sympathetic innervation to the pineal gland.

Pinealocyte synaptic ribbons (SR) and dense-cored vesicles (DCV) were quantitated in mice that have developmental deficiencies in the sympathetic innervation of the pineal gland due to a null mutation for the p75 low-affinity NGF receptor (NGFR). SR exhibited a significant increase whereas there was a significant decline in the frequency of DCV in mutant mice. These findings support the hypothesis that pinealocyte SR and DCV are regulated by neural mechanisms associated with sympathetic system. Ultrastructural characteristics of pinealocytes in mutants included degenerative changes that culminated in the breakdown of cellular components and the accumulation of flocculent-containing vesicles within the cytoplasm. Ribosomal lamellar complexes were also commonly seen in pinealocytes of mutant rats. Although the mutant pineal gland exhibited signs of metabolic imbalances, the cytoarchitecture of the gland (e.g., vascular compartment) and differentiation of the cells were generally unaffected by developmental deficiencies in the gland's innervation.

Increased recurrence and metastasis in patients whose primary head and neck squamous cell carcinomas secreted granulocyte-macrophage colony-stimulating factor and contained CD34+ natural suppressor cells.

Human head and neck squamous cell carcinomas (HNSCC) that produce high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) have been shown to contain CD34+ natural suppressor cells that inhibit the activity of intratumoral T-cells. The present study evaluated whether GM-CSF production and the presence of CD34+ cells within primary HNSCC would translate into increased recurrence, metastasis or cancer-related death during the 2 years following surgical excision. Freshly excised primary HNSCC of 20 patients that subsequently developed disease, and of 17 patients that remained with no evidence of disease were analyzed for production of GM-CSF and for CD34+ cell content. The cancers of patients that subsequently developed recurrences or metastatic disease produced almost 4-fold the levels of GM-CSF and had approximately 2.5-fold the number of CD34+ cells as did cancers of patients that remained disease-free. In a second method of analysis, the prognostic significance of high vs. low GM-CSF and CD34+ cell values was evaluated. These analyses showed that patients whose cancers produced high GM-CSF levels or had a high CD34+ cell content had a disproportionately high incidence of recurrence or metastatic disease (94% and 100%, respectively), while the majority of patients whose primary cancers produced low levels of GM-CSF or had a low CD34+ cell content remained disease-free (16% and 19%, respectively). Our results indicate that the presence of CD34+ cells in GM-CSF-producing HNSCC is associated with a poorer prognosis for the cancer patients and suggest the utility of these parameters as prognostic indicators of outcome. Mechanistically, our results suggest that the presence of immune suppressive CD34+ cells in GM-CSF-producing HNSCC leads to increased tumor recurrence or metastasis.

Increased interleukin-6 production by cerebral cortical tissue of adult versus young mice.

The cytokine interleukin-6 (IL-6) has been implicated as a contributor to injury in several neurological disorders. The amounts of IL-6 released by the cerebral cortical tissue of mice of varying maturational age groups were measured and found to increase with age. Specifically, the basal level of IL-6 released from the tissue of infant (less than 2 weeks old) mice was low, although the tissue could be readily stimulated to secrete high levels of IL-6. Higher levels of IL-6 were released from young adult (2 month old) mice. Adult mice that were either 6 or 9 months of age secreted significantly higher levels of IL-6 compared to the tissue of either the young adult or infant animals, although the difference between levels secreted by the 6- or 9-month old groups was minimal. IL-6 production by adult cerebral cortical tissue could be further stimulated, but it was less readily achieved compared to the tissue of infants. In response to the negative regulator transforming growth factor-beta (TGF-beta), the levels of IL-6 released by stimulated cerebral cortical tissue of infants, as well as by unstimulated and stimulated tissue of adults, were reduced to the low basal levels of IL-6 produced by infant tissue. These results suggest that normal development and aging are correlated with an increase in IL-6 production that may be due to shifts in levels of stimulatory or inhibitory regulatory controls, but not to an inability of young tissue to produce IL-6 or to a lack of responsiveness of adult tissue to negative regulatory control by TGF-beta.

Vitamin D3 treatment of tumor bearers can stimulate immune competence and reduce tumor growth when treatment coincides with a heightened presence of natural suppressor cells.

By secreting granulocyte-macrophage colony-stimulating factor (GM-CSF), Lewis lung carcinoma tumors induce immune suppressive granulocyte-macrophage progenitor cells. Treating mice having established tumors and high levels of suppressor activity with vitamin D3 eliminated suppressor activity, increased anti-tumor immunity, induced an immune stimulatory cell population, and reduced tumor growth. When instead, the vitamin D3 treatment was initiated earlier, when implanted tumors first became detectable and when natural suppressor activity was less prominent, the treatment had no effect. Thus, vitamin D3 treatment can stimulate the immune competence of tumor bearers when treatment is targeted to coincide with a heightened presence of GM-CSF-induced suppressor cells.

Mechanisms of immune suppression in patients with head and neck cancer: influence on the immune infiltrate of the cancer.

Freshly excised human head and neck cancers (219 primary cancers; 64 metastatic lymph node cancers) were analyzed for the immune inhibitory mediators released from the cancer tissues and the immune infiltrate within the tumor. Significant levels of the immune inhibitory mediators transforming growth factor-beta (TGF-beta), prostaglandin E2 (PGE2) and interleukin-10 (IL-10) were released from these cancers. Also released was granulocyte-macrophage colony-stimulating factor (GM-CSF), whose secretion was associated with an intratumoral presence of CD34+ cells. We have previously shown that CD34+ cells within human head and neck cancers are immune inhibitory granulocyte-macrophage progenitor cells. The presence of TGF-beta, PGE2 and IL-10 was associated with a reduced content of CD8+ T-cells within the cancers. The CD4+ cell content appeared to be less affected by these immune inhibitory mediators. Instead, parameters indicative of CD4+ cell function (p55 IL-2 receptor expression, release of IL-2 and IFN-gamma) were diminished in cancers that released higher levels of TGF-beta, IL-10 and GM-CSF and had a higher CD34+ cell content. Furthermore, metastatic cancers released higher levels of the soluble immune inhibitory mediators and lower levels of IFN-gamma and IL-2 than did primary cancers, although CD34+ cells were similarly present in both primary and metastatic cancers. Our results show that human head and neck cancers have a multiplicity of non-mutually exclusive mechanisms of immune suppression that are most prominently associated with reduced CD8+ cell influx and reduced influx and altered function of intratumoral CD4+ cells.

Immune modulation by interleukin-12 in tumor-bearing mice receiving vitamin D3 treatments to block induction of immunosuppressive granulocyte/macrophage progenitor cells.

Lewis lung carcinoma (LLC-LN7) tumors stimulate myelopoiesis and increase the presence of granulocyte/macrophage (GM) progenitor cells having natural suppressor activity. Treatment of these tumor-bearing mice with interleukin-12 (IL-12) resulted in minimal immune modulation. The objective of this study was to determine whether eliminating natural suppressor activity would allow for immune stimulation by IL-12. Treatment of LLC-LN7 tumor-bearing mice with vitamin D3 eliminated natural suppressor activity. In mice that were first treated with vitamin D3 and then also with IL-12, there was stimulation of splenic T cell proliferation in response to immobilized anti-CD3 plus IL-2. In addition, spleen and lymph node cells from vitamin-D3/IL-12-treated tumor-bearing mice became stimulated in response to autologous tumor to produce interferon gamma (IFN gamma), although IL-2 production was not stimulated. A prominent effect of the combined vitamin-D3/IL-12 treatment regimen was the synergistic augmentation of autologous tumor-specific cytolytic activity within the regional lymph nodes. The generation of these tumor-specific effector cells required the presence of the tumor mass since such activity was not elicited in the lymph nodes of mice from which the tumors had been surgically excised. The results of this study show that, after treatment of tumor bearers with vitamin D3 to eliminate GM-suppressor cells, IL-12 can induce select regional antitumor immune responses, particularly IFN gamma production and cytolysis by regional lymph node cells of autologous tumor.