Further characterization of cytotoxic T cells generated by short-term culture of human peripheral blood lymphocytes with interleukin-2 and anti-CD3 mAb

 In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3⁺ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fcγ receptor (FcγRI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab′)₂, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18⁺ ovarian tumor cell line), was also compared in the presence of a bispecific antibody (OC/TR, anti-CD3 × MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 ×antitumoral bispecific antibodies. Received: 6 December 1995 / Accepted: 4 June 1996     

Phase I trial of adoptive immunotherapy of cancer patients using monocyte-derived macrophages activated with interferon γ and lipopolysaccharide

 Cells of the monocyte/macrophage lineage have shown antitumor activity in vitro and in murine models after activation with interferon (IFN) γ. In vitro data suggest an additional effect on macrophage antitumor activity when IFNγ is combined with endotoxin (lipopolysaccharides; LPS). In this study we treated nine cancer patients with a total of 62 MAK infusion cycles with autologous macrophages given intravenously (i.v.) after in vitro activation with IFNγ and LPS. Low-grade fever (WHO I/II) was the commonest side-effect. Chills, nausea, and headache were noted when the number of transfused macrophages exceeded 2×10⁸. One WHO IV toxicity occurred, consisting of hypotension after transfer of 3×10⁸ cells, defining this dose as the maximum cell number tolerated. After pretreatment with ibuprofen, however, the maximum cell number could be increased without reaching dose-limiting toxicity. The highest number of cells reinfused was 15×10⁸. Circulating interleukin(IL)-6 increased in a dose-dependent manner as did IL-1 receptor antagonist (IL-1RA) and IL-8. Tumor response consisted of one case of stable disease (12 weeks) in a patient with formerly progressing colorectal cancer and progressive diseases in eight patients. This study indicates that reinfusion of autologous LPS-activated macrophages upon pretreatment with ibuprofen is feasible and tolerated without major side-effects. Received: 22 May 1997 / Accepted: 2 October 1997     

Evaluation of natural killer cell expansion and activation in vivo with daily subcutaneous low-dose interleukin-2 plus periodic intermediate-dose pulsing

 Natural killer (NK) cells may be expanded in vivo with a prolonged course of daily subcutaneous interleukin-2 (IL-2). However, cellular activation requires higher concentrations of IL-2 than are achieved with low-dose therapy. The objective of the current trial was to determine the toxicity and immunological effects of periodic subcutaneous intermediate-dose IL-2 pulses in patients receiving daily low-dose therapy. A group of 19 patients were treated with daily subcutaneous low-dose IL-2 at 1.25×10⁶ International Units (1.25 MIU) m^–2 day^–1. After 4–6 weeks, patients received escalating 3-day intermediate-dose IL-2 pulses administered as single daily subcutaneous injections, repeated at 2-week intervals. The maximum tolerated pulse dose was 15 MIU m^–2 day^–1, with transient hypotension, fatigue, and nausea/vomiting dose-limiting. Subcutaneous IL-2 resulted in in vivo expansion of CD56⁺ NK cells (796±210%) and CD56^bright natural killer (NK) cells (3247±1382%). Expanded NK cells coexpressed CD16, and showed lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity in vitro. Intermediate-dose pulsing resulted in serum IL-2 concentrations above 100 pM. Cellular activation was suggested by rapid margination of NK cells following pulsing, coincident with peak IL-2 levels, with return to baseline by 24 h. In addition, interferon γ production in response to lipopolysaccharide was augmented. Subcutaneous daily low-dose IL-2 with intermediate-dose pulsing is a well-tolerated outpatient regimen that results in in vivo expansion and potential activation of NK cells, with possible application in the treatment of malignancy and immunodeficiency. Received: 31 December 1997 / Accepted: 20 April 1998     

Differential modulation by interferon γ of the sensitivity of human melanoma cells to cytolytic T cell clones that recognize differentiation or progression antigens

 Human melanoma is a highly immunogenic tumor capable of inducing a specific immune response. A number of melanoma-associated antigens have been characterized during the past several years and can be classified into two groups: differentiation antigens  –  present also in normal melanocytes  –  and tumor-specific antigens, which, with the exception of testis, are present only in tumor cells. In a previous publication [Kirkin A. F., Petersen T. R., Olsen A. C., Li L., thor Straten P., Zeuthen J. (1995) Cancer Immunol Immunother 41:71] we have described the production of clones of cytotoxic T lymphocytes (CTL) against the highly immunogenic human melanoma cell line FM3. Using these clones we have defined four previously unknown melanoma-associated antigens, which could be subdivided into differentiation and progression antigens. In the experiments reported in this paper, we have further compared CTL clones from different groups and shown that the sensitivity of melanoma cells to CTL that recognize differentiation or progression antigens is differentially modulated during tumor progression as well as by the lymphokines interferon γ (IFNγ) and interleukin-10 (IL-10). The interaction of CTL clones recognizing progression antigens was strongly increased after treatment of melanoma cells with IFNγ, while the recognition by CTL clones specific for differentiation antigens either was unchanged or significantly decreased. IL-10 treatment of melanoma cells induced up-regulation with respect to recognition by CTL clones specific for differentiation antigens without affecting the recognition of melanoma cells by CTL clones specific for progression antigens. Using cellular systems at different stages of tumor progression, we demonstrated that the progressed state of melanoma cells is associated with increased sensitivity to recognition by CTL clones detecting progression antigens, and with decreased sensitivity to CTL clones recognizing differentiation antigens. Mimicking tumor progression, treatment with IFN-γ induced apparent down-regulation of differentiation antigens. A hypothesis is suggested in which IFN-γ plays different roles in the immune response against poorly immunogenic and highly immunogenic melanoma cells, increasing the progression of poorly immunogenic tumor cells or promoting a strong immune response and regression of highly immunogenic melanoma cells. Received: 23 November 1995 / Accepted: 7 March 1996     

Induction of accessory cell function of human alveolar macrophages by inhalation of human natural interleukin-2

 Accessory function allows antigen-presenting cells to produce sufficient secondary signals for optimum T cell proliferation and interleukin-2 (IL-2) production. Alveolar macrophages are inferior accessory cells compared to monocytes (PBM). We report here that the accessory index (AI) of alveolar macrophages and PBM of patients with lung metastases of solid tumors treated with inhalations of human natural IL-2 (hnIL-2) increased following its administration (P<0.005). The accessory index was significantly elevated from baseline values after 2 weeks of inhalation of 300 000 IU hnIL-2/day (8.2±10.2 compared to 1.1±1; P<0.001). The inhalation of 150 000 IU also induced increases in the index (AI = 2.3±1.9), however, without reaching statistical significance. In addition at 300 000 IU IL-2/day a significant increase in the accessory index was observed for PBM (4±2.5; P<0.05). The indices of PBM and alveolar macrophages prior to inhalation showed a significant negative correlation with the age of the patients (r _s =  – 0.5; r _s =  – 0.8, respectively; P<0.03 for all comparisons). Our data demonstrate that the inhalational application of hnIL-2 enhances the accessory function of alveolar macrophages and, to lesser extent, the accessory index of PBM, indicating the occurrence of pharmacological immunostimulation. Received: 16 August 1995 / Accepted: 4 January 1996     

Bacillus Calmette-Guérin plus interleukin-2 and/or granulocyte/macrophage-colony-stimulating factor enhances immunocompetent cell production of interferon-γ, which inhibits B16F10 melanoma cell growth in vitro

 Although immunotherapy with bacillus Calmette Guérin (BCG) is an established adjuvant treatment for malignant melanoma, the mechanism of its role in this process is unclear. To investigate the possible contribution of tumor-inhibitory cytokines induced by BCG, B16F10 melanoma cell growth in culture was assessed in response to purified cytokines and conditioned media of BCG-stimulated splenocytes. Interferon-γ (IFNγ) was the most potent single agent (IC₅₀≈50 pg/ml). Tumor necrosis factor α was substantially weaker (IC50>10 ng/ml) but provided synergy with IFNγ. None of the other cytokines such as interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-12, or granulocyte/macrophage-colony-stimulating factor had direct antitumor activity against B16F10 melanoma cells. However, when IL-2 and/or GM-CSF were combined with BCG either by exogenous addition or through endogenous production by novel cytokine-secreting recombinant BCG (rBCG), a substantial increase in INFγ production by splenocytes was observed. Antitumor activity of this conditioned medium directly correlated with IFNγ concentration and was completely blocked by neutralizing antibody to IFNγ. These results suggest that BCG may exert part of its antitumor action on melanoma through the induction of IFNγ, which can be greatly enhanced through the concomitant addition of IL-2 and/or GM-CSF. Furthermore, by utilizing rBCG that secrete these cytokines, it may be possible to potentiate the antitumor effect of BCG directly at the site of BCG inoculation. Received: 29 January 1996 / Accepted: 9 April 1996     

Interleukin-2 and interleukin-7 augment the cytolytic activity and expand the antitumor killing spectrum of α CD3-induced activated killer cells: potential use in the immunotherapy of non-immunogenic tumors

 This study investigates the effect of different cytokines on the growth and antitumor activity of the α CD3-induced killer cells CD3-AK, and the potential of the use of CD3-AK cells in cancer immunotherapy. Eight cytokines were tested. Only three (interleukin-2, -4 and -7) were able to support the growth of CD3-AK cells, which selectively killed different tumor targets of diversified origin. Culturing in interleukin-4 (IL-4) or IL-7 alone could maintain the growth of CD3-AK cells for 6 – 8 days. Only IL-2 could maintain long-term growth, but further addition of IL-4 exerted an inhibitory effect, which terminated the cell growth in 2 weeks. In contrast, despite the fact that IL-7 inhibited the proliferation of CD3-AK cells cultured in IL-2, as determined by [³H]thymidine uptake, the recovery of viable cells was not reduced. In 10 days, CD3-AK cells cultured in IL-2 alone or IL-2 plus IL-7 increased 160- or 176-fold respectively. There is an inverse relationship between the in vitro growth ability and Fas expression on the CD3-AK cells. Further, IL-7 increased the cytolytic activity of the CD3-AK cells two- to threefold. CD3-AK cells could be maintained in IL-2 or IL-2 plus IL-7 for 60 – 240 days or more. The long-term-cultured CD3-AK cells not only possessed a high level of cytolytic activity, but also showed a wide spectrum of killing with different tumor targets; the normally “resistant” targets, such as EL-4 lymphoma, fibrosarcoma, or melanoma, became susceptible. When the in vivo antitumor activity of the CD3-AK cells against a non-immunogenic tumor, EL-4, was tested by tumor-neutralization experiments, we found that only the long-term-cultured cells gave significant protection, with those maintained in both IL-2 and IL-7 giving the highest degree of protection. Thus, these long-term-cultured CD3-AK cells may have the potential to be used for immunotherapy of a variety of tumors whatever their immunogenicity. Received: 7 August 1996/accepted: 10 October 1996     

Intratumoral infusion of the monoclonal antibody, mAb 425, against the epidermal-growth-factor receptor in patients with advanced malignant glioma

 Malignant glioblastoma may over-express the epidermal-growth-factor receptor (EGF-R). Normal brain cells show a low or no expression of EGF-R. A mouse monoclonal antibody (IgG2A) (mAb 425) (EMD55900) (Merck KGaA, Bernstadt, Germany) directed against EGF-R was produced for therapeutic use. Eight patients with primary or recurrent, EGF-R-positive glioblastomas entered the study, which was designed to evaluate the clinical effect of the mAb. In order to achieve a high tumor cell saturation, the mAb was injected intratumorally twice weekly through an implantable catheter. The total administered dose varied between 4 mg and 120 mg. In 3 patients with solid tumors, a massive tumor necrosis was noted, with infiltration of macrophages, granulocytes and T cells. A further 3 patients developed clinical and radiological signs of an intense, local, inflammatory reaction. There may be a relation between the mAb dosage and the antitumor effect, insofar as higher doses seemed to cause a more pronounced, inflammatory reaction. Of the 8 patients, 6 developed human, anti-(mouse Ig) antibodies. This anti-EGF-R mAb may induce an intense, inflammatory reaction and a considerable necrosis in glioblastoma. However, the planned schedule could not be completed, even after the dose level was re-adjusted, owing to inflammatory reactions, which were severe without prior tumor debulking. Received: 12 November 1996 / Accepted: 3 March 1997     

Apoptosis in antibody-dependent monocyte-mediated cytotoxicity with monoclonal antibody 17-1A against human colorectal carcinoma cells: enhancement with interferon γ

 Antibody-dependent cell-mediated cytotoxicity (ADCC) has been considered to be one of the main effector mechanisms by which unconjugated monoclonal antibody (mAb) 17-1A can exert an antitumor effect in vivo. Since the apoptotic pathway as well as the necrotic pathway have been shown to be utilized in various cytotoxic effector mechanisms, we investigated the role of apoptosis in ADCC mediated by monocytes (ADMC) using mAb 17-1A as an antibody and the human colorectal carcinoma cell line, COLO205, as target cells in vitro. The implications of the apoptosis during ADMC was demonstrated by means of both a DNA fragmentation assay and a TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Furthermore, interferon γ (IFNγ) was also found to enhance the induction of apoptosis significantly. The addition of superoxide dismutase did not reduce the level of the apoptosis, although superoxide anion (O₂ ^–) was observed to be produced. However, the release of tumor necrosis factor α (TNFα) was significantly enhanced during ADMC, while, in addition, apoptosis was significantly inhibited by the addition of anti-TNFα antibody. These findings indicated that apoptosis might be implicated in ADMC with mAb 17-1A, which was augmented by IFNγ, while, in addition, TNFα may also be one of the major mediators of apoptosis. Received: 1 August 1996 / Accepted: 27 August 1996     

Tumor immunotherapy: cytokines and antigen presentation

 Increasing the ability of tumor-reactive T cells to mediate tumor regression in vivo has been a major goal of tumor immunologists. Progress toward this goal has been aided by the identification of tumor-associated antigens on both experimental mouse tumors and human tumors. However, the self-like nature and low immunogenicity of these antigens has made it clear that other measures to enhance the effectiveness of the T cells reactive to these antigens are essential if immunotherapy is to be clinically effective. An increased understanding of antigen processing and presentation is an important step in this process, as is the use of cytokines to increase immune responsiveness. Despite recent advances, there is still much to be learned before the specificity of the immune system is safely harnessed to halt malignant cell growth effectively. Received: 10 October 1997 / Accepted: 12 January 1998     

Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts: studies of allostimulated interferon-γ secretion

 Normal peripheral blood mononuclear cells (PBMC responders) were cultured together with non-irradiated allogeneic PBMC (more than 95% leukaemia blasts) derived from patients with acute leukaemia (referred to as leukaemic PBMC stimulators). Cytokine secretion was determined as cytokine concentrations in supernatants. Both normal PBMC and enriched CD4⁺ and CD8⁺ T cells responded to allostimulation with interferon (IFNγ) secretion. Interleukin-1 (IL-1) receptor antagonist and IL-2-neutralizing antibodies decreased IFNγ secretion. Exogenous IL-1β, IL-2 and IL-7 increased allostimulated IFNγ secretion, whereas decreased levels were seen in the presence of IL-6, IL-10 and granulocyte-colony-stimulating factor (G-CSF). During allorecognition IFNγ -neutralizing antibodies decreased acute myelogenous leukaemia (AML) blast secretion of G-CSF. We conclude that (i) both CD4⁺ and CD8⁺ T cells show allostimulated cytokine secretion in response to allogeneic stimulator cells containing a dominating population of native, cytokine-secreting leukaemia blasts, and (ii) IFNγ released during this response can modulate the function of allogeneic AML blasts. Received: 4 June 1996 / Accepted: 15 October 1996     

Taxol-mediated changes in fibrosarcoma-induced immune cell function: Modulation of antitumor activities

 The anticancer drug taxol (paclitaxel) inhibits tumors through multiple cytotoxic and cytostatic mechanisms. Independently of these mechanisms, taxol induces distinct immunological efficacy when it acts as a second signal for activation of tumoricidal activity by interferon-γ(IFNγ)-primed murine normal host macrophages. We reported that tumor-distal macrophages, which mediate immunosuppression through dysregulated nitric oxide (NO) and tumor necrosis factor α (TNFα) production, are differentially regulated by taxol. Because taxol influences tumor cell growth dynamics and activates immune cell populations, we assessed the ex vivo immunosuppressive and antitumor activities of taxol-treated normal host and tumor-bearing host (TBH) macrophages. Pretreatment of such cells with taxol partly reconstituted T cell alloantigen reactivity, suggesting that taxol mediates a limited reversal of TBH macrophage immunosuppressive activity. Taxol-treated TBH macrophages significantly suppressed the growth of fibrosarcoma cells (Meth-KDE) through soluble effector molecules and promoted direct cell-mediated cytotoxicity, indicating that taxol enhanced tumor-induced macrophage antitumor activities. Tumor-induced helper T cells, however, showed a higher sensitivity to direct taxol-induced suppression. These data demonstrate that taxol exerts pleiotropic effects on antitumor immune responses with the capacity to abate the immunosuppressive activities of macrophages and promote macrophage-mediated antitumor activities simultaneously, but also directly modulating T cell reactivity. Collectively, these studies suggest that the antineoplastic drug taxol may impart antitumor activity through an immunotherapeutic capacity. Received: 31 December 1996 / Accepted: 1 July 1997     

Early-appearing tumor-infiltrating natural killer cells play an important role in the nitric oxide production of tumor-associated macrophages through their interferon production

 Both natural killer (NK) cells and macrophages are thought to be the main effectors responsible for early antitumor defense. In this study, we investigated the role of tumor-infiltrating NK cells in initiating nitric oxide (NO) production by tumor-associated macrophages (TAM). The in vivo depletion of NK cells prior to the i.p. inoculation of melanoma cells resulted in a significant decrease in the NO production of the TAM prepared from the peritoneal exudate cells (PEC). Such prior NK cell depletion also decreased the ability of TAM to show any antitumor activity in vitro. The addition of N ^G-monomethyl-L-arginine (Me-L-Arg) to the culture partially inhibited the ability of TAM to suppress the proliferation of melanoma cells and also decreased their cytolytic activity against melanoma cells. These results suggest that the TAM exhibited both cytostatic and cytolytic activities through their NO production. In an in vivo assay, the administration of Me-L-Arg permitted the more rapid growth of i.p. inoculated melanoma cells compared with the control. On the other hand, the decreased NO production of TAM, resulting from the prior NK cell depletion, was restored by the i.p. administration of interferon γ (IFNγ). In addition, the in vivo administration of anti-IFNγ mAb into mice inoculated i.p. with melanoma cells also significantly decreased the NO production of TAM in peritoneal exudate cells. Furthermore, the tumor-infiltrating NK cells produced a considerable level of IFNγ. Overall, these results indicate that early-appearing tumor-infiltrating NK cells play an important role in the NO production of TAM through their IFNγ production. Received: 11 March 1997 / Accepted: 31 July 1997     

Immune response against medullary thyroid carcinoma (MTC) induced by parental and/or interleukin-2-secreting MTC cells in a rat model of human familial medullary thyroid carcinoma

 The existence of inherited aggressive forms of medullary thyroid carcinoma (MTC), and their resistance to all classical therapies, make it a prime candidate for adoptive immunotherapy. As a prelude to a vaccine for the protection of family members at risk of developing the disease, we investigated the immunological antitumour response provoked by the 6/23 rMTC cell line, compared to that of the same cells engineered to secrete interleukin-2 (rMTC-IL2), in an animal model of familial human MTC, the inbred strain of Wag/Rij rats. The rMTC cells developed a tumour that invaded the whole neck 15 days after orthotopic injection (into the thyroid), while the rMTC-IL2 cells were progressively rejected. Co-injection of rMTC-IL2 with the parental cells induced the rejection of the rMTC transplants. When injected, both tumoral cell types showed a similar positive immunoreaction with anti-MHC class I (major histocompatibility complex class I) antibodies. They both recruited natural killer cells and eosinophils at the site of injection. In addition, CD8⁺ T lymphocytes infiltrated the rMTC-IL2 cells, and eosinophil recruitment was amplified. Neutrophils, macrophages and CD4⁺ T lymphocytes were scarce. Our results suggest that the CD8⁺ T lymphocytes are implicated in the antitumour reaction elicited by the Il-2-transfected cells. As these effectors are known to induce a specific immunological response, including memory, such a protocol should be tested as a vaccine on the young population genetically at risk of developing a MTC. Received: 18 December 1995 / Accepted: 21 August 1996     

Analysis of Th1 and Th2 cytokine production by peripheral blood mononuclear cells as a parameter of immunological dysfunction in advanced cancer patients

Purpose: The presence of immunological dysfunction has not been well demonstrated in cancer patients. Recent studies have revealed that the immune response can be classified into types 1 and 2, and in the present work the immunological function of patients was studied from the perspective of these two types of response. Methods: Types 1 and 2 immune response were evaluated by monitoring the production of various cytokines by peripheral blood mononuclear cells from 38 patients with advanced cancer of various organs and 20 healthy subjects. The usual immunological parameters, differential cell leukocyte counts, the level of T cell subsets (CD4 and CD8) and natural killer activity were also examined. Results: The production of interleukin-2 (IL-2), interferon γ, IL-10, IL-12 and tumor necrosis factor α was found to be significantly lower in the patients (75 ± 57, 171 ± 205, 40 ± 34, 8 ± 8, 1450 ± 1010 pg/ml) than in healthy subjects (143 ± 99, 422 ± 296, 64 ± 34, 16 ± 10, 2550 ± 950 pg/ml); however, the mean level of IL-4 in the patients seemed to be higher. The correlations between different cytokine levels suggested that they were produced differently. Lymphocyte counts were significantly lower in patients, but there was no difference in the other usual immunological parameters. Conclusions: Patients with advanced cancer are deficient in monocytes and the type 1 immune response. The measurement of various cytokines reported in this study provides a more sensitive and valuable tool for evaluating the function of cell-mediated immunity in cancer patients than do the usual tests. Received: 10 March 1999 / Accepted: 24 June 1999     

Nitric oxide inhibits proliferation but increases life-span of T lymphocytes in tumour-bearing rats

 Nitric oxide (NO) has been shown to inhibit the proliferation of lymphocytes. However, in tumour-bearing rats treated with the immunomodulator OM 163, the regressing nodules were heavily infiltrated by T lymphocytes, although they contained high levels of NO. We show here that NO, while inhibiting the proliferation of lymphocytes, increased their life-span, pointing to the ambivalence of this molecule in the course of tumour growth and regression. Received: 16 October 1997 / Accepted: 8 January 1998     

Cellular immune responses to autologous chronic myelogenous leukaemia cells in vitro

 Using a modification of the autologous mixed lymphocyte/tumour cell culture (MLTC), it is demonstrated here that lymphocytes from chronic-phase myelogenous leukaemia (CML) patients (n = 58), but not from their HLA-identical siblings, proliferated upon coculture with autologous tumour cells. However, in most cases, the level of proliferation measured was low (stimulation index <3, n = 37). This was most likely related to the amount of interleukin-10 (IL-10) released into the culture medium by the CML cells, because addition of neutralizing anti-IL-10 serum to MLTC markedly enhanced proliferative responses. In addition, supplementation of media with IL-1α further enhanced proliferative responses and a combination of anti-IL-10 serum and IL-1α was more effective than either agent alone. Only HLA-DR-matched CML cells, but not HLA-DR-mismatched CML cells or matched or mismatched PBMC restimulated proliferation of IL-2-dependent T cell lines derived from MLTC supplemented with IL-1α and anti-IL-10 serum. The responding cells under these conditions were predominantly CD4⁺ and secreted IL-2, and interferon γ; some secreted IL-4, but none secreted IL-10. These data therefore suggest the existence of an HLA-DR-restricted DTH/Th1-type of tumour-specific immunity in CML patients, which may be down-regulated in vitro by excessive secretion of IL-10 together with depressed secretion of IL-1. Received: 9 November 1995 / Accepted: 8 February 1996     

Introduction of the interferon γ gene into mouse T lymphoma cells with low MHC class I-expression results in selective induction of H-2Dk and concomitant enhanced metastasis

 Interferon-γ(IFNγ)-induced up-regulation of MHC class I expression on tumor cells can induce a potent CD8-mediated antitumor response. Consequently, many investigators have proposed IFNγ gene transfection as a means to immunogenize tumor cells and to vaccinate against metastatic disease. In this study, we demonstrate that transfection of the IFNγ gene in a BW5147 variant (LiD^lo) with low MHC class I expression results in a selective induction of H-2D^k but unaltered H-2K^k expression. In earlier reports we demonstrated a positive correlation between H-2D^k expression and enhanced metastatic potential of BW variants. In accordance with these observations, we observed that intravenous inoculation of LiD^lo(IFNγ) variants into syngeneic AKR mice led to enhanced metastasis as compared to parental LiD^lo and LiD^lo(neo) control transfectants. Tumor cells, derived from local subcutaneous tumors or sporadic metastases from mice inoculated with LiD^lo tumor cells, were found to up-regulate H-2D^k selectively. Anti-asialoGM1 treatment of AKR mice allowed rapid experimental metastasis formation by the LiD^lo and LiD^lo(neo) variants, indicating that natural killer (NK) cells control the metastatic behavior of these tumor cells. This was corroborated by in vitro cytotoxicity experiments, demonstrating that LiD^lo and LiD^lo(neo) tumor cells were NK-sensitive, while the BW IFNγ transfectants became resistant to lymphokine-activated killer cells and poly(I)·poly(C)-induced NK cells. We thus conclude that (a) IFNγ up-regulates selectively the MHC class I antigen H-2D^k, (b) H-2D^k governs susceptibility towards NK cells, and (c) NK susceptibility determines the experimental metastatic behavior of BW tumor cells. Received: 2 May 1996/Accepted: 21 May 1996     

The prognostic significance of immune changes in patients with renal cancer,melanoma and colorectal cancer,treated with interferon α2b

 In the present study we evaluated the response rate and the immunorestorative properties of interferon α2b (IFnα2b) administered to patients with advanced renal cell carcinoma (RCC), melanoma (MEL) or colorectal cancer (CC). We studied the immune status and correlated it with clinical responses. Thirty-five patients with advanced RCC, and 14 with MEL were treated with recombinant INFα2b. The dose was increased progressively from 5×10⁶ IU/day in the first week (three times every week) to 10×10⁶ IU/day in the second week and thereafter to 15×10⁶ IU/day subcutaneously. In patients with CC INFα2b was given at 5×10⁶ IU/day every other day (three times every week); these patients also received (together with INF) leucovorin 200 mg m^–2 day^–1 in a 1-h i. v. infusion every week, and mid-infusion 400 mg/m² 5-FU was administered as an intravenous bolus every week. The response rate was as follows: for RCC, 6 patients achieved partial response (PR), 10 stable disease (SD), and 21 progressed (PD); for MEL, 5 patients achieved PR and 9 PD; for CC, 6 achieved PR, 5 SD, and 9 PD. In all patients blood was withdrawn prior to INFα2b treatment and then monthly. T lymphocytes, after isolation from peripheral blood, were tested for proliferation in the autologous mixed-lymphocyte reaction and allogeneic mixed-lymphocyte reaction, interleukin-2 (IL-2) production, expression of IL-2 receptors during the allogeneic-mixed-lymphocyte reaction, and the production of IL-1 by peripheral blood monocytes. Striking increases were demonstrated in all parameters 2 months after treatment with INFα2b. In comparison to normal controls, all patients with the malignant neoplasms presented decreased (>45%) mean values of the immunological parameters under investigation (P 0.0001). Responders (patients with RCC, MEL, and PR) presented lower mean values of all the parameters studied than did non-responders (P 0.0001). Patients with CC presented the lowest mean values of the parameters than did the other patients (RCC, MEL) (P 0.0001). After therapy with INFα2b, patients with RCC experiencing PR showed a mean increase of more than 30% (P 0.0001). Patients with SD showed a mean increase of about 20% (P 0.0001), and those with PD showed a 6% increase in the immunological parameters under investigation. Patients with MEL experiencing PR showed a mean increase of more than 30% and patients with PD a decrease of more than 10% (P 0.0001). All patients, regardless of the clinical response, achieved an increase of more than 60% (P 0.0001). Administration of IFNα2b resulted in a marked potentiation of a deficient cellular immune response in vitro in those patients with RCC and MEL who responded to the treatment. On the other hand, non-responders demonstrated a decrease in the examined parameters and, in some, deterioration of the already depressed immunological functions was observed. This observation can have prognostic significance regarding clinical response of INF. In contrast, our findings show that the immune stimulation associated with INFα treatment in all our CC patients did not predict an improved clinical outcome. There are several theoretical explanations for this discrepancy. Received: 30 November 1996 / Accepted: 25 June 1996